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LABORATORY  DIRECTIONS 


FOR    BEGINNERS    IN 


BACTERIOLOGY 


AN   INTRODUCTION  TO   PRACTICAL   BACTERIOLOGY 
FOR  STUDENTS  AND  PRACTITIONERS  OF  COM- 
PARATIVE  AND   OF   HUMAN   MEDICINE 


BY 

VERANUS   A.  MOORE,  B.S.,  M.D. 

Professor  of  Comparative  Pathology  and  Bacteriology 
Cornell  University,  Ithaca,  N.Y. 


THIRD  EDITION 
ENLARGED  AND  REVISED 


GINN  &  COMPANY 

BOSTON  .  NEW  YORK  •  CHICAGO  •  LONDON 

,.K.. 


Copyright,  1898,  1900,  1905,  by 
VERANUS  A.  MOORE 


ALL    RIGHTS    RESERVED 


r    -giV\'n'&.c;Oivi-pa!iy  ■-  r Rp- 

-^    PR^ETGlvS^.  B0ST6M  •  USA. 


f-\^osl 


PREFACE  TO   THE  THIRD   EDITION 

The  rapid  advances  that  are  constantly  being  made  in 
bacteriological  work  demand  frequent  changes  in  directions 
for  laboratory  practice.  The  subject-matter  contained  in  this 
elementary  work  has  been  readjusted  to  satisfy  these  require- 
ments, a  few  exercises  on  new  topics  have  been  introduced, 
and  the  methods  given  have  been  modified  to  better  meet  the 
conditions  of  student  laboratory  work.  Continued  experience 
has  strengthened  our  former  opinion  that  a  laboratory  guide 
which  outlines  the  work  for  each  exercise  and  gives  specific 
instructions  for  the  same  is  of  great  assistance  to  both  student 
and  teacher.  I  desire  to  express  my  thanks  and  appreciation 
to  Mr.  G.  Franklin  White  and  Mr.  Walter  E.  King  for  many 
valuable  suggestions  that  have  been  incorporated  in  the  recast- 
ing of  these  exercises.  Y   ^   j^ 

Ithaca,  N.Y. 
November,  1904 


2ii5 


PREFACE  TO   THE  SECOND   EDITION 

The  call  for  a  second  edition  of  these  Laboratory  Direc- 
tions has  come  in  such  a  short  time  that  many  of  the  diffi- 
culties encountered  in  the  preparation  of  the  first  edition  still 
remain.  The  choice  of  subject-matter  and  the  selection  of 
methods  for  a  short  elementary  laboratory  course  become 
more  and  more  difficult  with  the  rapidly  increasing  develop- 
ments in  bacteriology.  The  recognized  etiological  importance 
of  a  number  of  bacteria  which  formerly  were  considered  of 
little  significance  necessitates,  for  the  best  results,  an  extension 
of  a  knowledge  of  bacteriology  beyond  the  differential  charac- 
ters and  properties  of  a  few  pronounced  pathogenic  species. 

Experience  with  the  first  edition  has  clearly  demonstrated 
the  advantage  to  both  student  and  teacher  of  specific  direc- 
tions for  a  working  basis  in  carrying  out  the  various  procedures 
in  a  laboratory  course.  The  exercises  have  been  considerably 
modified,  four  new  ones  added,  and  a  few  references  appended 
for  the  purpose  of  aiding  students  in  familiarizing  themselves 
with  the  current  literature  on  the  subject. 

In  revising  these  exercises  new  text  and  reference  books 
have  been  freely  consulted.  Valuable  suggestions  have  also 
been  received  from  a  number  of  teachers  and  investigators.  I 
am  especially  indebted  for  such  assistance  to  Dr.  Theobald 
Smith  of  Harvard  University,  Dr.  Erwin  F.  Smith  of  the  United 


VI  PREFACE  TO  THE   SECOND    EDITION 

States  Department  of  Agriculture,  and  to  Mr.  Raymond  C. 
Reed  and  Mr.  Floyd  R.  Wright,  Instructors  in  the  Department 
of  Bacteriology  in  Cornell  University.  Suggestions  and  criti- 
cisms which  may  tend  to  increase  the  usefulness  of  these 
outlines  are  cordially  invited. 

V.  A.  M. 
Ithaca,  N.Y. 
June,  1900 


PREFACE  TO   THE   FIRST   EDITION 

It  has  been  found  desirable  to  provide  the  student,  just 
beginning  the  study  of  bacteriology,  with  a  somewhat  detailed 
outhne  of  the  work  to  be  done  at  each  laboratory  session. 
The  selecting  of  the  particular  things  to  be  done  and  the 
choosing  of  methods  to  be  followed  are  difficult  tasks.  The 
assigning  of  directions  for  doing  work  under  assumed  condi- 
tions must  necessarily  partake  of  the  empirical,  and  often  fail. 
It  is  evident,  however,  that  practical  bacteriology  must,  if 
successfully  taught,  be  cast  in  a  somewhat  definite  form  in 
order  that  the  student  may  come  to  a  knowledge  of  the  funda- 
mental principles  underlying  the  subject  in  its  twofold  capacity, 
that  of  a  pure  science  and  of  a  useful  art. 

These  outHnes  are  intended  either  to  serve  simply  as  a 
guide  through  an  introductory  laboratory  course  preparatory 
to  independent  research  work,  or  to  form  the  basis  for  the 
application  of  the  principles  of  bacteriology  in  the  practice 
of  human  or  of  comparative  medicine.  They  aim  to  impart 
a  technical  and  working  knowledge  of  certain  of  the  more 
essential  methods  and  to  develop  a  definite  knowledge  of  a 
few  important  species  of  bacteria.  During  the  past  year,  they 
were  furnished  the  students  in  mimeographed  sheets,  but  after 
making  the  changes  suggested  by  this  application  it  seems 
desirable  to  put  them  in  a  more  convenient  form.     In  adjust- 


VIU  PREFACE  TO  THE   FIRST  EDITION 

ing  the  amount  of  work  for  each  exercise  to  the  necessary 
limitations  of  time  and  facilities,  I  am  indebted  to  Mr.  Ray- 
mond C.  Reed,  Instructor  in  this  Department,  for  much 
valuable  assistance.  I  wish  also  to  thank  Professor  Charles 
Wright  Dodge  of  the  University  of  Rochester  for  helpful 
suggestions.  Should  these  outlines  fall  in  the  hands  of  other 
teachers  or  workers  in  this  subject,  criticisms  are  cordially 
invited. 

V.  A.  M. 
Ithaca,  N.Y. 
August,  1898 


CONTENTS 


PAGE 

List  of  Text  and  Reference  Books xiii 

Apparatus  and  Material xvii 

Laboratory  Maxims xix 

Introduction xxi 

EXERCISES 

I.     Cleaning  glassware i 

II.     Plugging  tubes  and  flasks  and  sterilizing  the  glassware .  5 

III.  The  preparation  of  bouillon 7 

IV.  The  preparation  of  gelatin  and  agar        .         .         .         .11 
V.     Inoculating  tubes  of  bouillon,  agar,  and  gelatin      .         .  14 

VI.     The  examination  of  cultures 17 

VII.     Making  and  staining  cover-glass  preparations,  and  for- 
mulae for  staining  solutions 23 

VIII.     Making  plate  and  Esmarch  roll  cultures          ...  29 
IX.     The  examination  of  plate  cultures  and  the  making  of 

subcultures  from  colonies 32 

X.     The  preparation  of  certain  differential  and  special  media  39 

XI.     Inoculating  special  media  and  examining  cultures  .         .  46 

XII.     The  examination  of  cultures  on  special  media        .         .  48 

XIII.  The  examination  of  cultures  (continued)         .         .         -52 

XIV.  The  classification  of  bacteria -53 

XV.     The   morphology   of   streptococcus,   micrococcus,   and 

sarcina 56 

XVI.     The  morphology  of  bacillus 58 

XVII.     The  morphology  of  bacterium  and  spirillum  ...  59 
XVIII.     Staining  spores         .         .         .         .         .         .         .         .60 

XIX.     Staining  the  flagella  on  motile  bacteria  ....  62 

XX.     Staining  tubercle  bacteria  (bacilli) 67 

XXI.     A  study  of  certain  saprophytic  bacteria  ....  69 

XXII.     A  study  of  bacteria  in  milk 71 

XXIII.     A  study  of  bacteria  in  water 72 

XXIV.     A  study  of  certain  pyogenic  bacteria       •         •         •         •  73 

ix 


CONTENTS 


or  Micrococcus 


or  Micrococcus 


EXERCISES  PAGE 

XXV.  Pyogenic  bacteria  (continued) 74 

XXVI.  Pseiidomonas  pyocyaneus  ......       75 

XXVII.  Bacillus  coli  communis 76 

XXVIII.  Bacillus  coli  communis  and  the  paracolon  .         .       78 

XXIX.  Bacillus  cholerce  suis  and  Bacillus  typhosus       .         .       79 

XXX.  Bacillus  cholerce  stiis  Sind  Bacilltts  typ/iosus  (continued)    81 

XXXI.  Bacillus  cholerce  suis  and  Bacilhis  typhosus  (continued)    83 

XXXII.  Bacilli  of  dysentery    . 

XXXIII.  Widal  serum  test       . 

XXXIV.  Bacterium  septiccemice  hemorrhagicce 

lanceolatus 

XXXV.  Bacterium  septiccemice  hemorrhagicce 

lanceolatus  (continued) . 

XXXVI.  Bacterium  tuberculosis 

XXXVII.  Bacterium^  mallei 

XXXVIII.  Bacterium  mallei  (continued) 

XXXIX.  Cultures  of  anaerobic  bacteria 

XL.  Bacillus  tetani   . 

XLI.  Bacterium  anthracis . 

XLII.  Bacterium  anthracis  (continued) 

XLIII.  Bacterium  diphtherice 

XLIV.  Bacterium  diphtherice  (continued) 

XLV.  The  bacteria  of  the  healthy  mouth 

XLVI.  Identifying  bacteria  from  cultures 

XLVII.  Isolating  and  identifying  bacteria  from  animal  tissues 

XLVIII.  Isolating  and  identifying  bacteria  from  animal  tissues 

(continued) 

XLIX.  The   examination    of   sections  of  tissue  containing 

bacteria  

L.  Bacteriologic  examination  of  pus  and  exudates 

LI.  A  bacteriologic  examination  of  the  skin  for  Micro- 
coccus epidermidis  albus  and  other  bacteria    . 

LII.  Determining  the  thermal  death  point  of  bacteria 

LIII.  Determining  the  efficiency  of  disinfectants 

LIV.  Pasteurizing  and  steriUzing  milk         .... 

LV.  The  quantitative  bacteriologic  examination  of  water 

LVI.  The  qualitative  examination  of  water 

LVII.  Examination  of  certain  bacteria  not  studied  in  the 

laboratory,  pathogenic  fungi  and  protozoa     . 

LVIII.  Bacteriological  diagnosis    ...... 


85 
88 

90 
91 
94 

95 

96 

98 

100 

lOI 

102 
104 
105 
107 
108 


III 
112 

114 
116 
118 
120 
122 
124 


126 
127 


CONTENTS 


XI 


APPENDIX 


I.  Reaction  of  culture  media     .... 

II.  The  ocular  micrometer  and  micrometry 

III.  Animal  inoculation  for  purposes  of  diagnosis 

IV.  Cultivation  of  Bacterium  {Bacillus)  tuberculosis 
V.  Jeffers'  plate  and  metric  system    . 


PAGB 
140 

147 


Index 


149 


A  LIST  OF  THE  MORE  IMPORTANT  TEXT  AND 
REFERENCE  BOOKS 

"^  Abbott,  A.  C.    The  Principles  of  Bacteriology.    New  6th  edition.    1902. 
V  Abel,  R.     Taschenbuch  fiir  den  bakterienlogischen,  u.  s.  w. 
^  Archinard  and  Pedersen.     Microscopy  and  Bacteriology.     1903. 
f  Baumgarten.     Jahresbericht  u.  d.  Fortsch.  d.  path.  Mikroorganismen. 

BowHiLL,  Thos.     Manual  of  Bacteriological  Technique  and  Special 
Bacteriology.     1899. 

Chester,  F.  D.      A  Manual  of  Determinative  Bacteriology.     1901. 

Conn,  H.  W.     Bacteria  in  Milk  and  its  Products.     1903. 

Crookshank,  E.  M.     Text-Book  of  Bacteriology  and  Infectious  Dis- 
eases.    1897. 

Curtis,  H.  J.     The  Essentials  of  Practical  Bacteriology.     1900. 

Doyen  and  Roussel.     Atlas  de  Microbiologie.     1897. 

EiSENBERG,  J.     Bacteriological  Diagnosis.    From  2d  German  edition. 
1892. 

Emery,  W.  D.     Handbook  of  Bacteriological  Diagnosis.     1902. 

Eyre,  J.  W.  H.     The  Elements  of  Bacteriological  Technique.    1902. 

Fischer,  A.     The  Structure  and  Functions  of  Bacteria.     1900. 

Flugge,  C.     Die  Mikroorganismen.      1896. 

Fraenkel,  C.     Text-Book  of  Bacteriology.     3d  edition.     1891. 

Fraenkel  and  Pfeiffer.     Mikrophotographischer  Atlas  der  Bakte- 
rienkunde.     1893. 

Frankland,  p.  and  G.  C.     Micro-organisms  in  Water. 

Frost,  W.  D.     Laboratory  Guide  in  Bacteriology.     1901. 

Gedoelst,  L.     Traite  de  Microbiologie.     1899. 

Heim,  L.     Lehrbuch  der  bakteriologischen  Untersuchung  und  Diag- 
nostik.     1894. 

Hewlett,  R.  T.     A  Manual  of  Bacteriology.     1902. 

HuEPPE,  F.     The  Principles  of  Bacteriology.     1899. 

Jorgensen,  A.     Micro-organisms  and  Fermentation.    3d  edition.    1900. 

Lehmann  and  Neumann.     Atlas  and  Principles  of  Bacteriology.    1901. 

Mac]6,  E.     Traite  Pratique  de  Bacteriologie.     1901. 

Mosselman  and  Lienaux.    Manual  of  Veterinary  Microbiology.    1894. 


XIV  REFERENCE   BOOKS 

McFarland,  J.     A  Text-Book  upon  the  Pathogenic  Bacteria.     4th 

edition.     1903. 
MiGULA,  W.     System  der  Bakterien.     1900. 

MuiR  and  Ritchie.   Manual  of  Bacteriology.   American  edition.    1903. 
Newman,  G.     Bacteria.     1899. 

Now,  F.  G.     Laboratory  Work  in  Bacteriology.     2d  edition.     1899. 
Park,  W.  H.     Bacteriology  in  Medicine  and  Surgery.     1899. 
Pearmain  and  Moor.     Applied  Bacteriology.     1898. 
Sternberg,  George  M.  Text-Book  of  Bacteriology.    2d  edition.    1901. 
Sternberg,  George  M.    A  Manual  of  Bacteriology.     1892. 
SwiTHiNBANK  and  Newman.     Bacteriology  of  Milk,     1903. 
Thoinot  and  Masselin.     Precis  de  Microbie.     1896. 
Williams,  H.  U.     A  Manual  of  Bacteriology.     3d  edition.     1903. 
WooDHEAD,  G.  S.     Bacteria  and  their  Products.     1897. 
WuRTZ,  R.     Precis  de  Bacteriologie  Clinique.     1897. 


JOURNALS  AND  PERIODICALS  OF  SPECIAL  VALUE 
TO  THE  STUDENT  OF  BACTERIOLOGY 

Centralblatt  fiir  Bakteriologie,  Parasitenkunde  u.  Infektionskrankheiten. 

The  Journal  of  Pathology  and  Bacteriology. 

Zeitschrift  fiir  Hygiene  u.  Infectionskrankheiten. 

Annales  de  I'lnstitut  Pasteur. 

Archives  de  Med.  Experimentale  et  d' Anatomic  Pathologique. 

The  Journal  of  Experimental  Medicine. 

The  Journal  of  Medical  Research. 

The  Journal  of  Infectious  Diseases. 

Annual  Reports  of  the  American  Public  Health  Association. 

All  standard  medical  and  veterinary  journals. 

Important  articles  on  various  topics  in  bacteriology  frequently  appear 
in  journals  on  sanitary  engineering,  botany,  chemistry,  general  biology, 
reports  of  city  and  state  Boards  of  Health,  United  States  Government 
Reports  (especially  .those  of  the  Bureau  of  Animal  Industry  and  of  the 
Marine  Hospital  Service),  State  Experiment  Station  Bulletins,  and 
reports  of  scientific  societies. 


BOOKS   VALUABLE   FOR  METHODS   AND   FORMULAE 

Von  Kahlden.     Methods  of  Pathological  Histology. 

Lee.     The  Microtomist's  Vade-Mecum. 

Lafar.     Technical  Mycology. 

Saccardo.     Chromotaxia  seu  Nomenclator  Colorum.     Patavii,  1894. 

Gage.     The  Microscope. 

Mallory  and  Wright.     Pathological  Technique. 


APPARATUS  AND   MATERIAL 

Apparatus.  The  apparatus  and  material  for  use  in  bacteri- 
ology in  a  student  laboratory  fall  very  naturally  into  three 
groups,  viz.  :  ( i )  apparatus  to  be  used  in  common  by  all  stu- 
dents, and  for  which  no  individual  is  responsible  excepting 
when  in  actual  use  by  him,  and  the  supplies  from  which  each 
student  draws  the  necessary  quantity  for  the  work  assigned ; 
(2)  apparatus  to  be  assigned  to  each  student  for  personal 
use  and  for  which  he  is  wholly  responsible ;  and  (3 )  material 
such  as  notebooks  to  be  provided  by  each.  The  assignment 
of  equipment  and  supplies  in  accordance  with  this  plan  has 
been  made  in  a  general  way,  as  indicated  in  the  following 
paragraphs. 

(i)  Apparatus  in  laboratory  for  general  use.  This  includes 
the  apparatus  and  chemicals  to  be  used  in  common  by  all 
students,  and  consists  of  pans  and  brushes  for  cleaning  test 
tubes  and  other  glassware,  meat  mincer  and  press,  large  and 
small  water  baths,  steam  sterilizers,  hot-air  sterilizers,  incu- 
bators, thermometers,  thermostats,  gas  burners,  balances,  level- 
ing tripods,  Wolffhiigels'  or  other  apparatus  for  aid  in  counting 
colonies,  micrometers,  metric  rules,  burettes,  tripods,  funnels, 
beakers,  pipettes,  graduates,  glass  tubing  and  rods,  also  the 
chemicals  necessary  for  carrying  on  the  work,  such  as  various 
acids  and  alkalies,  disinfectants,  alcohol,  aniline  dyes,  and  those 
articles  needed  in  the  preparation  of  culture  media,  such  as  salt, 
peptone,  agar,  gelatin,  meat  extract,  sugars,  litmus  and  other 


XVlll  APPARATUS   AND  MATERIAL 

indicators,  and  filter  paper ;  fresh  meat,  eggs,  milk,  and  potatoes 
being  furnished  as  needed.  The  equipment  also  includes  color 
charts  and  the  more  important  books  of  reference. 

(2)  Apparatus  furnished  for  individual  use.  The  various 
appliances  used  by  each  student  and  for  which  he  becomes 
personally  responsible  are  a  microscope  with  substage  con- 
denser, two  oculars  (i  and  2  inch)  and  three  objectives 
(§,  ^,  and  y^  inch),  a  bottle  of  immersion  oil,  a  hand  mag- 
nifier, 75  small  test  tubes,  30  large  test  tubes,  10  fermentation 
tubes,  1 8  Petri  dishes,  3  Erlenmeyer  flasks,  7  one-ounce  bottles 
for  reagents  and  stains,  supplied  with  pipettes  or  glass  rods, 
I  platinum-wire  loop,  i  platinum-wire  needle,  3  tin  cups  for 
holding  cultures,  3  wire  baskets  for  holding  test  tubes,  i  block 
for  holding  reagent  bottles,  i  glass  slide  with  ring  attached 
for  hanging-drop  preparations,  i  tin  tray  for  cover-glass  prep- 
arations, 2  solid  watch  glasses,  2  ointment  jars  for  used  slides 
and  cover  glasses,  and  a  glass  box  for  clean  cover  glasses. 
Each  working  table  is  provided  with  a  reserve-flame  gas  burner 
(Bunsen),  glass  jars  for  waste,  and  stands  for  holding  culture 
tubes.  Requisite  amounts  of  absorbent  cotton,  lens  paper, 
and  towels  are  furnished  when  needed. 

(3)  Material  to  be  provided  by  each  student.  A  box  of  slides 
and  cover  glasses  (cover  glasses  f  inch  square  preferred ;  they 
must  be  between  .12  and  .18  mm.  in  thickness),  a  slide  box 
for  permanent  preparations,  gummed  labels,  preferably  with 
name  printed  upon  them,  for  slides  and  cultures,  a  Faber's 
blue  pencil  for  marking  on  glass,  fine  forceps  for  handling 
cover  glasses,  and  paper  for  laboratory  notes  with  manila 
cardboard  covers  or  suitable  notebooks. 


LABORATORY  MAXIMS 

1.  See  that  the  working  table,  instruments,  and  all  pieces 
of  apparatus  used  are  thoroughly  cleaned  at  the  close  of  each 
exercise. 

2.  Unless  otherwise  directed,  all  cultures,  other  than  those 
in  gelatin,  are  to  be  grown  in  the  incubator. 

3.  Gelatin  cultures  should  not  be  put  into  the  incubator 
except  for  special  purposes  not  described  in  these  direc- 
tions. 

4.  In  opening  tubes  of  media  or  cultures  always  flame  the 
open  end  of  the  tube  immediately  after  withdrawing  the  plug. 
If  the  tubes  have  been  standing  for  some  time,  the  surface 
of  the  plug  should  be  flamed  before  drawing  it  out.  Never 
allow  the  tube  end  of  the  plug  to  touch,  while  out  of  the  tube, 
any  article  by  which  it  could  become  contaminated.  It  should 
be  held  by  the  top  between  the  fingers. 

5.  In  making  transfers  the  tubes  should  be  held  as  nearly 
in  the  horizontal  position  as  possible.  Cultures  should  not  be 
opened  in  currents  of  air. 

6.  In  every  case  where  a  platinum-wire  loop  or  needle  is 
used  for  making  cultures  or  withdrawing  media  it  should  be 
carefully  heated  in  a  gas  flame,  or  that  of  an  alcohol  lamp, 
both  immediately  before  and  after  using.  The  heated  wire 
must  be  allowed  to  cool  before  making  cultures. 

7.  In  making  plate  cultures  work  as  much  as  possible  under 
a  hood  and  in  still  air. 


XX  LABORATORY  MAXIMS 

8.  If  by  accident,  a  drop  or  more  of  a  culture  should  be 
spilled  upon  the  table  or  floor,  pour  over  it  a  sufficient  quan- 
tity of  a  disinfectant  (corrosive  sublimate  solution  i-iooo,  or  a 
5  %  solution  of  carbolic  acid)  to  completely  cover  the  infected 
area.  After  this  has  acted  for  ten  minutes  wipe  it  up  and  boil 
or  burn  the  cotton  or  cloth.  If  any  of  the  culture  should  drop 
on  the  hands  or  clothing,  a  disinfectant  should  be  applied 
immediately. 

9.  In  sterilizing  culture  media,  always  see  that  there  is 
enough  water  in  the  pan  of  the  steam  sterilizer  or  in  the  water 
bath  before  Hghting  the  gas.  Do  not  put  the  media  in  a 
sterilizer  and  leave  the  laboratory. 

10.  Always  disinfect,  by  boiling,  all  cultures  before  cleaning 
the  tubes  or  plates  containing  them.  (A  liberal  supply  of 
cleaning  mixture  can  be  used  to  advantage  in  some  instances 
for  destroying  cultures.) 

11.  At  the  beginning  of  each  laboratory  session  read  the 
directions  for  the  next  exercise  in  order  to  be  able  to  make 
any  preliminary  preparations  which  may  be  required. 

12.  Careful  notes  should  be  taken  on  all  observations  made 
in  the  study  of  cultures  and  preparations  made  from  them. 


INTRODUCTION 

Plan  of  exercises.  Bacteriology  has  become  one  of  the 
recognized  branches  in  the  curriculum  of  all  medical  and  vet- 
erinary colleges.  In  many  universities  it  is  taught  as  a  part  of 
the  course  in  general  biology.  It  is,  however,  still  a  young 
science  and  the  best  methods  for  teaching  it  have  not  as  yet 
been  determined.  All  are  agreed,  however,  that  in  addition 
to  such  text-book  work  and  lectures  as  may  be  required  there 
should  be  laboratory  practice  in  actually  handling  and  study- 
ing various  bacteria  and  in  determining  their  special  morpho- 
logical characters  and  physiological  properties. 

In  order  to  differentiate  the  various  species  of  bacteria,  to 
isolate  them  from  impure  cultures  or  animal  tissues,  it  is  neces- 
sary that  one  should  be  familiar  with  the  methods  to  be  used ; 
otherwise  the  attention  will  be  directed  more  to  the  modus 
operandi  than  to  the  organisms  themselves.  On  this  point, 
however,  there  is  much  difference  of  opinion.  Some  labora- 
tory workers  believe  that  the  methods  should  be  taken  up  and 
learned  in  connection  with  the  study  of  the  various  species  of 
bacteria  and  thus  avoid  the  loss  of  time  that  special  instruc- 
tion in  methods  requires.  Others  favor  a  devotion  of  a  por- 
tion of  the  time  to  a  consideration  and  drill  in  the  methods 
to  be  employed  later  on  in  the  course  in  the  serious  study  of 
species  and  in  diagnostic  work. 


XXll  INTRODUCTION 

The  experience  in  this  laboratory  has  been  that  the  best 
results  are  obtained  by  teaching  the  more  fundamental  prin- 
ciples and  methods  as  such  before  attempting  to  apply  them 
in  the  study  of  the  various  species  of  bacteria  or  in  practical 
diagnostic  work.  It  has  happened,  even  when  the  number 
of  exercises  is  very  limited,  that  a  preliminary  drill  in  the 
methods  is  greatly  to  the  advantage  of  the  student.  From 
the  nature  of  the  subject,  its  application  can  be  made  and 
benefit  derived  therefrom  only  by  those  who  know  how  to  do 
the  things  that  the  exigencies  of  the  moment  demand.  This 
means  efficiency  in  knowing  how.  In  following  these  direc- 
tions, therefore,  the  student  must  understand  that  the  purpose 
of  the  first  twenty  exercises  is  to  teach  him  how  to  do  the 
things  called  for  in  the  later  exercises  in  the  study  of  species 
and  in  some  of  the  practical  applications  of  bacteriology. 

Another  feature  of  these  directions  is  that  they  aim  to  teach 
the  student  how  to  study  and  observe  bacteria  in  their  cul- 
tures rather  than  to  tell  him  what  he  is  to  observe.  It  is  not 
intended  that  they  should  take  the  place  of  lectures  and  text- 
books in  bacteriology.  Their  mission  is  to  aid  the  student  in 
finding  out  for  himself  what  the  text-books  relate  concerning 
certain  species,  and  to  guide  him  in  the  elementary  steps  in 
the  more  important  diagnostic  procedures,  tests,  and  analyses. 

The  fact  must  also  be  recognized  that  in  a  short  elementary 
course  it  is  not  possible  to  try  several  methods  for  doing  the 
same  thing.  This  restricts  us  to  the  use  of  a  single  pro- 
cedure. The  one  is  given  that  seems  to  us  best  adapted  to 
the  limited  time  and  facilities  of  the  student.  It  may  happen, 
however,  that  other  methods  would  be  preferable  under  other 


INTRODUCTION  XXlll 

conditions  or  in  the  hands  of  certain  individuals.  This  limi- 
tation is  a  necessity,  however,  in  an  elementary  course  of 
instruction.  Its  objection  is  partially  met  by  references  to 
text-books  and  other  publications  where  other  methods  are 
described.  It  is  very  important  that  the  student  familiarize 
himself  with  at  least  a  few  of  the  more  important  books  and 
periodicals  dealing  with  bacteriology.  They  are  the  source 
to  which  he  must  go  later  for  information  on  this  subject, 
and  consequently  a  knowledge  of  their  nature  and  how  to  use 
them  may  be  of  unmeasured  value. 


LABORATORY  BACTERIOLOGY 

EXERCISE  I 

CLEANING  GLASSWARE 

1.  It  is  necessary  that  the  glassware  employed  should  be 
thoroughly  cleaned  before  it  is  used.  Several  special  methods 
have  been  suggested  for  this  purpose,  but  the  one  frequently 
employed  by  chemists  seems  to  be  the  most  easily  handled 
and  quite  as  efficient  for  general  use  as  the  more  elaborate, 
specialized  processes.  It  consists  in  applying  the  chromic 
acid  cleaning  mixture  after  washing  the  tubes  and  flasks  with 
water.  Experience  has  taught  that  usually  this  method  may 
be  abandoned,  as  a  strong  alkali  is  quite  sufficient  to  clean 
the  ordinary  glassware.  It  is  sometimes  necessary  to  employ 
more  special  methods  for  cover  glasses  which  are  to  be  used 
in  staining  bacteria  where  a  mordant  is  required.  Only  one 
of  these  special  methods  will  be  given  here. 

2.  Work  for  this  exercise.  Clean  all  the  glassware,  test 
tubes,  fermentation  tubes,  flasks,  Petri  dishes,  and  reagent 
bottles  assigned. 

If  the  next  laboratory  period  occurs  on  the  following  day, 
place  the  flasks  and  test  tubes  in  the  incubator  to  insure  their 
being  dry  before  they  are  plugged. 

Put  the  slides  and  cover  glasses  in  the  cleaning  mixture ; 
they  can  be  rinsed  and  wiped  later. 

Read  the  laboratory  maxims. 

3.  Methods  to  be  followed  in  cleaning  the  different  appa- 
ratus, (a)  Test  tubes.  Wash  the  tubes  carefully  with  a  strong 
alkali  soap  and  water,  using  the  test-tube  brush.     Rinse  the 

I 


2  LABORATORY  BACTERIOLOGY 

tubes  thoroughly  in  hot  water  and  drain  them,  using  a  drain- 
age board.  [It  is  sometimes  desirable  to  use  the  cleaning 
mixture.-^  In  this  case,  after  the  tubes  are  washed  with  soap 
and  water,  they  are  stood  in  a  glass  jar  (aquarium)  and  filled 
with  the  cleaning  mixture.  After  it  has  acted  for  from  lo  to 
20  minutes,  pour  it  back  from  the  tubes  into  the  bottle  origi- 
nally containing  it.  The  tubes  are  then  to  be  thoroughly  rinsed 
and  dried  as  before.] 

(b)  Fer?nentation  tubes.  Treat  these  with  the  cleaning  mix- 
ture in  the  same  manner  as  the  test  tubes.  This  is  necessary 
because  the  brush  cannot  be  used. 

{c)  Flasks.  Wash  the  flasks  thoroughly  with  soap  and 
water.  Then  fill  them  with  the  cleaning  mixture  and  allow 
it  to  act  for  at  least  10  minutes,  after  which  it  is  to  be  poured 
back.  Rinse  the  flasks  thoroughly  in  the  same  manner  as  the 
test  tubes  and  drain  them.  When  dry  the  outside  should  be 
wiped  with  a  damp  cloth. 

(d)  Petri  dishes  and  reagent  bottles.  Thoroughly  wash  the 
Petri  dishes  and  reagent  bottles  in  hot  soapsuds,  after  which 
rinse  them  separately  in  tap  water  and  drain.  The  cleaning 
mixture  need  not  be  used.  After  they  are  dry  the  two  parts 
of  the  Petri  dish  should  be  put  together. 

{e)  Cover  glasses  and  slides.  Drop  the  cover  glasses  singly 
into  a  glass  jar  containing  cleaning  mixture  and  allow  them  to 
remain  there  for  24  hours  or  longer.  Pour  off  the  cleaning 
mixture  and  rinse  the  cover  glasses  in  boiled  water  until  all  the 
color  disappears ;  then  cover  them  with  alcohol  until  needed, 
when  they  can  be  wiped  with  a  soft  linen  cloth  or  with  lens 

^  Formula  for  chromic  acid  cleaning  mixture.  Dissolve  80  grams  of 
potassium  dichromate  (K2Cr207)  in  300  cc.  of  warm  water;  when  all  of 
the  K2Cr207  is  dissolved  and  the  solution  cooled,  add  it  slowly,  with 
constant  stirring,  to  460  cc.  of  concentrated  sulphuric  acid.  Store  the 
mixture  in  a  glass-stoppered  bottle.  The  liquid  will  be  quite  thick  with 
small  crystals.  When  the  crystals  are  used  up  the  liquid  should  be 
discarded. 


CLEANING  GLASSWARE  3 

paper.  Sometimes  there  appears  to  be  a  film  on  the  surface 
of  the  cover  glasses  which  interferes  in  making  hanging-drop 
preparations.  Sometimes  this  can  be  overcome,  after  they  are 
wiped  out  of  the  alcohol,  by  placing  them  in  a  Petri  dish  with- 
out the  cover  and  heating  them  in  the  dry-air  sterilizer  at  a 
temperature  of  i6o°  to  i8o°  C.  for  one  hour.  After  they  have 
cooled,  replace  the  cover  and  allow  the  cover  glasses  to  remain 
in  the  Petri  dish  (a  glass  jar  or  other  closed  dish  may  be  used) 
until  used.  (When  a  drop  of  water  or  bouillon  is  spread  upon 
a  properly  cleansed  cover  glass  it  does  not  roll  up  in  droplets, 
but  wil^  remain  in  a  thin,  even  layer  on  the  surface.) 

Slides  can  be  cleaned  very  satisfactorily  by  washing  them 
in  hot  soapsuds,  rinsing  them  in  hot  water,  and  wiping  them 
with  a  soft  cloth. 

(/)  Cleaning  used  culture  apparatus.  Place  the  tubes,  flasks, 
or  Petri  dishes  containing  old  cultures  in  a  water  bath,  cover 
them  with  water,  add  a  little  sal  soda  (about  an  ounce  to  a 
gallon  of  water),  and  boil  for  20  minutes.  Pour  off  the  water 
and  empty  the  tubes,  after  which  again  boil  them  for  5  minutes 
in  clean  soap  and  water.  Then  wash  and  treat  with  the  clean- 
ing mixture  the  same  as  the  new  tubes.  Cultures  of  spore- 
bearing  pathogenic  bacteria,  such  as  those  of  anthrax,  should 
be  destroyed  by  heating  in  the  autoclave  at  a  temperature  of 
at  least  110°  C.  for  half  an  hour  before  the  tubes  are  emptied 
and  washed. 

4.  A  method  for  cleaning  cover  glasses  for  flagella  stain. 
For  this  work  the  ordinary  method  of  cleaning  cover  glasses  is 
not  sufficient,  although  the  heating  will  often  give  a  perfectly 
satisfactory  cover  glass.  The  following  treatment  was  first 
highly  recommended  to  me  by  Dr.  Erwin  F.  Smith,  and  later 
modified  by  Johnston  and  Mack.  First  clean  the  cover  glasses 
by  the  ordinary  method,  after  which  boil  them  in  an  agate  cup 
or  glass  beaker  in  a  5  %  solution  of  caustic  soda  for  10  minutes. 
After  cooling,  rinse  thoroughly  in  distilled  water,  place  in  a 
beaker,  and  cover  with  a  10^  solution  of  hydrochloric  acid 


4  LABORATORY  BACTERIOLOGY 

and  boil  for  lo  minutes.  Then  pour  off  the  acid  and  rinse 
the  cover  glasses,  one  at  a  time,  using  forceps  for  handling, 
in  distilled  water  and  finally  in  ether-alcohol.  When  needed, 
wipe  with  a  piece  of  cheese  cloth  that  has  been  properly 
prepared  by  soaking  in  a  5%  solution  of  caustic  soda  for  a 
few  hours,  then  rinsed  in  water,  and  then  placed  for  a  few 
hours  in  dilute  (10%)  hydrochloric  acid,  after  which  it  should 
be  thoroughly  rinsed  in  distilled  water  and  dried.  Before 
wiping  the  covers  cleanse  the  hands  thoroughly  in  soap  and 
water,  then  in  ether. 

5.  Laboratory  notes.  In  this  and  all  subsequent  exercises 
careful  notes  should  be  taken  on  the  work  done.  When,  how- 
ever, as  in  this  exercise,  it  consists  simply  of  carrying  out 
directions,  a  simple  statement  that  the  work  as  directed  was 
completely  or  partially  done,  as  the  case  may  be,  is  all  that  is 
necessary.  In  all  other  cases  describe  fully  the  work  per- 
formed and  observations  made. 

The  notes  should  be  as  brief  as  completeness  will  permit. 
They  should  be  legibly  written  and  the  technical  terms  peculiar 
to  the  subject  in  hand  should  be  correctly  used.  The  notes  are 
to  be  handed  to  the  instructor  each  week  for  examination  and 
correction.  When  returned,  all  corrections  should  be  carefully 
noted  and  similar  errors  should  be  avoided  thereafter. 


PLUGGING  THE  TUBES  AND  FLASKS 


EXERCISE  II 

PLUGGING  THE  TUBES  AND  FLASKS  AND  STERILIZING 
THE  GLASSWARE 

6.  After  the  tubes  and  flasks  are  cleaned  they  must  be 
plugged.  The  plugged  tubes  and  flasks  and  the  Petri  dishes, 
all  of  which  are  to  be  used  for  holding  culture  media  or  in 
making  cultures,  must  be  sterilized  before  they  can  be  used. 
The  plugs  should  be  neatly  made  and  of  the  proper  length 
and  firmness.  The  best  quality  of  absorbent  cotton  is  ordi- 
narily used  for  this  purpose,  although  common  cotton  is 
employed  in  some  laboratories.  If  the  latter  is  used,  it  should 
be  first  heated  to  a  very  slight  browning  in  the  hot-air  ster- 
ilizer. This  drives  off  the  oil  and  kills  the  spores  which  it 
might  contain.  Glassware  is  sterilized  with  dry  steam  or  by 
means  of  dry  heat,  i.e.  in  the  hot-air  sterilizer.  (See  methods 
for  sterilizing  apparatus  and  instruments  in  text-books.) 

7.  Work  for  this  exercise.  Plug  all  the  tubes  and  flasks 
with  absorbent  cotton  and  sterilize  them,  together  with  the 
Petri  dishes. 

After  they  are  sterilized,  store  them  in  the  locker  until  they 
are  needed.  The  Petri  dishes  must  not  be  opened  until  they 
are  used. 

If  the  periods  are  short  this  exercise  and  the  following  one 
may  be  worked  together. 

8.  Plugging  the  tubes  and  flasks.  For  this  purpose  absorb- 
ent cotton  is  used.  Rolls  of  it  are  cut  in  short  segments 
of  from  5  to  7  cm.  in  length.  A  piece  of  this  narrow  strip 
of  sufficient  length  to  give  cotton  enough  for  the  plug  is 
torn  off.  The  quantity  varies,  of  course,  with  the  size  of 
the  mouth  of  the  tube  or  flask,  but  a  little  experience  will 
enable  one  to  estimate  the  quantity  quite  accurately.  The 
edges  of  the  piece  of  cotton  torn  off  are  turned  in  and  it 


6  LABORATORY  BACTERIOLOGY 

is  rolled  up  to  form  a  firm  plug  which  should  snugly  fit  the 
neck  of  the  tube  or  flask.  It  should  be  inserted  into  the  tube 
for  about  2  cm.  and  the  end  should  be  nearly  flat  and  smooth. 
The  projecting  part  should  be  about  the  same  length  and 
of  equal  firmness.  (For  method  of  closing  the  tubes  more 
securely,  see  §  22.) 

9.  Sterilizing  glassware,  (a)  Hot  air.  Place  all  the  test 
tubes,  flasks,  and  Petri  dishes  in  the  hot-air  sterilizer,  close  the 
door  tightly,  and  light  the  gas.  Heat  the  air  in  the  sterilizer 
to  a  temperature  of  from  135°  to  150°  C.  and  keep  it  there 
for  one  hour,  not  allowing  it  to  rise  above  150°  C.  Turn 
the  gas  oif ,  and  when  the  temperature  of  the  air  in  the  ster- 
ilizer goes  down  to  or  below  45°  C.  the  door  may  be  opened 
and  the  apparatus  removed. 

(b)  Dry  stea7n,  Sterihze  the  fermentation  tubes  in  the  auto- 
clave at  15  lbs.  pressure  for  half  an  hour.  This  method  pre- 
vents a  considerable  amount  of  breakage,  especially  of  the 
stand  of  the  tube,  that  often  occurs  when  these  tubes  are 
heated  in  the  hot-air  sterilizer. 


THE   PREPARATION   OF  BOUILLON  7 

EXERCISE  III 

THE   PREPARATION  OF  BOUILLON 

10.  Bouillon  is  the  liquid  medium  most  commonly  employed 
in  cultivating  bacteria.  It  is  practically  a  beef  tea  containing 
peptone.  There  are  several  methods  recommended  for  mak- 
ing it.  It  may  be  made  directly  from  simple  meat  infusion 
or  it  may  be  made  from  meat  extract.  The  meat  infusion 
is  prepared  either  by  allowing  finely  chopped  lean  meat  mixed 
with  twice  its  quantity  of  distilled  or  filtered  and  boiled  tap 
water  (2  cc.  of  water  for  each  gram  of  meat)  to  stand  in  a 
cool  place  for  from  12  to  18  hours,  or  the  mixture  of  meat 
and  water  may  be  heated  with  frequent  stirring  at  a  tempera- 
ture of  65°  C.  for  a  short  time  (one  hour).  Each  has  its 
advantages.  When  meat  extract  is  used  in  place  of  the  meat 
infusion,  the  bouillon  does  not  seem  to  be  a  favorable  culture 
fluid  for  certain  bacteria.  In  making  bouillon,  therefore,  it 
becomes  necessary  to  determine  the  kind  (whether  from  meat 
infusion  or  extract)  and  the  method  of  preparing  it  to  suit  the 
conditions  in  hand.  It  is  sometimes  desirable  in  bacteriologic 
investigations  to  resort  to  all  of  these  methods.  For  routine 
work  in  the  laboratory,  bouillon  prepared  directly  from  the 
meat  by  macerating  it  for  a  short  time  at  a  high  temperature 
(60°  to  65°  C.)  is  very  satisfactory.  The  addition  of  peptone 
and  the  neutralization  of  the  Hquid  are  the  same  in  both  cases. 

Bouillon  is  used  as  the  nutritive  base  in  preparing  agar  and 
gelatin.  On  this  account  large  quantities  are  stored  in  flasks. 
(For  other  methods  see  text-books.  Also  Report  (Journal)  of 
the  Am.  Public  Health  Asso.,  January,"  1898,  p.  77.) 

References.  Chapters  on  making  culture  media  in  text- 
books. Jour,  of  the  Am.  Public  Health  Asso.,  October,  1895,  and 
January,  1898,  p.  ^^^  Smith,  The  Jour,  of  Exper.  Med.,  Vol.  Ill 
(1898),  p.  647. 


8  LABORATORY  BACTERIOLOGY 

11.  Work  for  this  exercise.  Make  looo  cc.  of  bouillon  and 
distribute  it  as  follows  : 

Put  5  cc.  in  each  of  lo  small  sterile  test  tubes. 

Put  300  cc.  in  each  of  2  large  (500  cc.)  Erlenmeyer  flasks, 
and  the  remainder  in  a  third  flask. 

Put  5  cc.  of  distilled  water  in  each  of  5  small  sterile  test 
tubes  and  sterilize  them  with  the  bouillon.  (They  are  to  be 
used  subsequently  in  place  of  bouillon  in  making  dilutions.) 
Label  the  tubes  *'  sterile  distilled  water."  All  media  required 
to  carry  out  the  directions  will  be  furnished  by  the  instructor 
excepting  such  as  the  student  is  directed  to  make  in  Exercises 
III,  IV,  and  X. 

12.  The  preparation  of  bouillon.  Take  500  grams  of  lean 
beef,  remove  all  fat,  and  grind  it  in  a  sausage  machine  or  have 
it  minced  at  the  butcher  shop.  Place  the  minced  meat  in  an 
agate-iron  dish,  add  1000  cc.  (2  parts  water  to  one  of  meat) 
of  clear  boiled  water,  cooled  to  65°  C,  and  stir  thoroughly 
with  a  glass  rod.  Then  macerate  it,  with  frequent  stirring,  in 
a  water  bath  at  a  temperature  of  60°  C.  for  one  hour  after  the 
temperature  of  the  meat  and  water  reaches  that  of  the  water 
outside  or,  to  save  time,  at  a  temperature  of  65°  C.  for  30 
minutes.  Remove  the  meat  by  straining  the  liquid  through  a 
piece  of  cheese  cloth.  For  this  a  stout  iron  meat  press  is 
desirable.  Boil  the  strained  meat  infusion  for  20  minutes.^  Cool 
to  harden  the  fat,  and  filter  through  filter  paper.  The  filtrate 
should  equal  in  quantity  the  amount  of  water  used ;  if  it  does 
not,  add  enough  distilled  or  clear  boiled  water  to  make  up  that 
amount.  To  this  meat  infusion  add  1%  peptone  (Witte's) 
and  \f^o  sodium  chloride.  Add  enough  of  a  normal  solution 
of  sodium  hydrate  to  give  the  liquid  a  faintly  alkaline  reaction 
when  litmus  is  used  as  an  indicator.  (For  method  of  titrating 
media,  see  §  14.)     The  infusion  is  then  boiled  in  a  water  bath 

1  In  case  of  a  short  period,  the  exercise  may  be  divided  at  this  point 
by  boiling  the  infusion  for  30  minutes  and  keeping  it  overnight.  The 
receptacle  should  be  covered. 


THE   PREPARATION  OF  BOUILLON  9 

for  three  quarters  of  an  hour  and  filtered  hot.  The  filtrate 
should  be  perfectly  clear.  The  color  will  vary  according  to  the 
amount  of  blood  pigment  in  the  meat  used,  and  according  to 
the  length  of  time  it  is  steamed  or  boiled,  i.e.  according  to  the 
amount  of  material  precipitated  out.  After  filtering,  distribute 
the  bouillon  in  tubes  and  flasks  (see  above),  and  stand  them  in 
a  wire  basket  for  steriHzation.  Sterilize  them  by  boiling  in  a 
closed  water  bath  or  steaming  in  the  Arnold's  steam  sterilizer  for 
30  minutes,^  the  time  to  be  computed  from  the  time  the  water 
boils  or  the  temperature  in  the  steamer  reaches  99*^.  The 
flasks  of  bouillon  should  be  boiled  or  steamed  for  20  minutes 
on  each  of  the  two  succeeding  days  (certain  anaerobic  bac- 
teria may  not  be  destroyed  by  this  treatment).  When  they 
have  cooled  the  outside  of  the  tubes  should  be  carefully  wiped 
with  a  moist  cloth  and  placed  in  the  incubator  until  the  next 
laboratory  day.  Then  carefully  examine  them,  and  if  any  of 
the  tubes  are  contaminated,  that  is,  if  the  liquid  is  clouded  or 
has  a  membrane  on  the  surface,  they  must  be  rejected.  Label 
the  others  and  place  them  in  the  locker. 

1  The  customary  method  of  sterilizing  culture  media  is  to  steam  or 
boil  them  for  about  10  minutes  on  each  of  3  consecutive  days.  This 
was  found  very  troublesome  by  the  students,  and,  feeling  that  it  was  not 
necessary,  a  long  series  of  test  experiments  was  made  by  Mr.  R.  C.  Reed, 
who  found  that  i  boiling  or  steaming  for  30  minutes  gave  just  as  good 
results  as  the  customary  3  boilings.  As  the  media  are  not  used  for  2  or 
3  days  after  sterilization,  during  which  time  they  are  kept  in  an  incu- 
bator, the  method  is  well  suited  to  student  laboratories,  not  for  the 
reason  that  it  saves  time  in  preparing  the  media,  but  it  relieves  the 
congestion  in  the  sterilizer  and  appreciably  aids  the  student.  When 
sterilized  by  this  method  the  media  must  not  be  inoculated  for  several 
days  after  their  preparation  or  ufitil  they  have  stood  in  aii  i7tctibator  for 
at  least  18  hours  to  test  their  sterility. 

Media  can  be  quickly  sterilized  by  means  of  the  autoclave  when  the 
temperature  is  raised  from  110°  to  115°  C.  While  this  method  is  quick 
and  convenient,  the  high  temperature  seems  to  be  detrimental  to  media 
for  certain  pathogenic  bacteria.  The  autoclave,  however,  is  quite 
extensively  used. 


lO  LABORATORY   BACTERIOLOGY 

13.  Labeling  media  and  culture  tubes.  Stick  on  each  tube 
of  medium,  about  3  cm.  from  the  top,  an  adhesive  white  label 
about  2  cm.  square.  On  the  upper  line  should  be  written  the 
name  of  the  medium  and  the  date  of  its  preparation.  Thus, 
"  bouillon,  13-VII-1900."  When  the  tube  is  used  the  name  of 
the  organism  or  material  with  which  it  is  inoculated,  together 
with  the  date  of  inoculation,  should  be  written  on  the  lower 
lines.     This  applies  to  all  media  and  tube  or  flask  cultures. 

14.  Titration  of  bouillon.  Take  5  cc.  of  bouillon  and  place 
in  a  porcelain  evaporating  dish  with  about  45  cc.  of  water.  Boil 
for  three  minutes.  Add  i  cc.  of  a  solution  of  phenol-phthalein. 
Stir  and  add  to  the  solution  in  the  evaporating  dish  enough 
of  a  N/20  NaOH  solution  from  a  burette  to  give  it  a  clear, 
bright  pink  color.  This  amount  then  of  N/20  NaOH  solution 
is  required  to  neutralize  5  cc.  of  the  bouillon.  A  provisional 
standard  reaction  of  + 1 .5  to  phenol-phthalein  has  been  adopted. 
This  would  be  slightly  alkaline  to  litmus.  In  order  to  bring 
the  entire  amount  of  bouillon  to  the  desired  reaction  (-f-  1.5), 
subtract  1.5  from  the  amount  of  N/20  NaOH  solution  drawn 
from  the  burette  and  multiply  the  difference  by  the  number 
of  cc.  of  bouillon  divided  by  100.  The  product,  if  plus,  repre- 
sents the  amount  of  N/i  NaOH  to  be  added;  if  minus,  the 
amount  of  N/i  HCL  to  be  added.  After  mixing,  test  the 
reaction  again  in  the  same  way  and  add  alkali  or  acid  if  needed. 
For  greater  accuracy  further  tests  should  be  made.  (For  a 
more  complete  discussion  of  the  reaction  of  culture  media,  see 
Appendix  I.) 


THE   PREPARATION   OF  GELATIN  AND  AGAR        II 

EXERCISE   IV 

THE  PREPARATION  OF  GELATIN  AND  AGAR 

15.  Of  the  solid  media  employed  in  cultivating  bacteria,  agar 
and  gelatin  are  most  commonly  used.  They  depend  for  their 
nutritive  properties  largely  upon  the  bouillon  from  which  they 
are  made,  the  agar  and  gelatin  forming  simply  the  solidifying 
elements.  The  striking  difference  between  the  two  is  that  the 
gelatin  melts  at  the  body  temperature,  whereas  the  agar  is  not 
quickly  hquefied  below  the  boiling  point.  For  this  reason 
gelatin  is  not  used  as  a  solid  medium  for  cultivating  bacteria 
at  a  high  (body)  temperature.  There  are  several  processes  for 
preparing  these  media,  but  the  addition  of  the  dry  agar  and 
gelatin  to  bouillon  (§  12)  either  immediately  after  it  is  filtered 
or  later  after  it  has  been  sterilized  and  stored  in  flasks  seems 
to  be  the  most  convenient  procedure.  The  agar  itself  is  usu- 
ally neutral  in  reaction,  but  the  gelatin  often  has  a  decidedly 
acid  reaction.  This  necessitates  the  careful  testing  of  the 
reaction  of  the  two  media,  although  the  bouillon  is  neutral  or 
slightly  alkaline. 

References.  Chapters  on  culture  media  in  text-books  on 
bacteriology.  The  preparation  of  nutritive  agar,  American  Micro- 
scopic Journal,  May,  1890.  A  rapid  method  of  making  agar-agar, 
Johns  Hopkins  Hospital  Bulletin,  No.  24,  July-August,  1892.  Jour, 
of  the  Am.  Public  Health  Asso.,  January,  1898. 

16.  Work  for  this  exercise.  See  that  bouillon  made  in 
Exercise  III  is  properly  sterilized.  Prepare  300  cc.  of  gelatin 
and  300  cc.  of  agar,  i.e.  start  with  300  cc.  of  bouillon  for  each. 
There  will  be  considerable  shrinkage  owing  to  the  amount  lost 
on  the  dishes,  filter,  etc.,  so  that  the  quantities  of  media  will  be 
appreciably  less  than  this  amount.  Distribute  each  medium  as 
follows  : 


12  LABORATORY   BACTERIOLOGY 

Put  5  cc.  in  each  of  lo  small  sterile  test  tubes. 

Put  lo  cc.  in  each  of  12  large  sterile  test  tubes. 

Put  the  remainder  in  the  flask  which  contained  the  bouillon. 

17.  The  preparation  of  nutrient  gelatin.  Take  a  flask  of 
bouillon  containing  300  cc.  and  pour  it  into  a  small  agate  iron 
dish,  add  30  grams  of  sheet  gelatin,  and  heat,  with  frequent 
stirring,  in  a  water  bath,  until  the  gelatin  is  dissolved.  Allow 
it  to  cool  to  a  temperature  between  45°  and  50°  C.  and  then 
add  the  white  of  one  egg  and  mix  it  thoroughly  by  stirring, 
or,  better,  by  pouring  the  gelatin  many  times  from  one  flask 
or  beaker  to  another.  After  the  egg  albumen  is  completely 
diffused,  return  the  liquid  gelatin  to  the  large  covered  water 
bath  and  boil  until  the  egg  albumen  is  firmly  coagulated. 
This  takes  about  20  minutes.  It  is  now  ready  for  filtering, 
which  must  be  done  while  the  gelatin  is  hot.  Filter  through 
properly  folded  ^  but  ordinary  filter  paper,  first  moistened  with 
boiling  water.  Distribute  the  filtrate  as  directed.  In  pouring 
the  gelatin  into  the  tube  use  a  small  beaker  or  graduate,  and 
see  that  the  gelatin  does  not  touch  the  sides  of  the  upper  part 
of  the  tube.  Stand  the  tubes  in  a  wire  basket  and  sterilize  them 
by  boiling  in  a  closed  water  bath  or  by  steaming  in  the  Arnold 
steam  sterilizer  for  30  minutes.  The  small  flasks  can  be 
sterilized  in  the  same  manner.  Place  tubes  and  small  flasks  in 
the  incubator  and  allow  them  to  remain  there  for  two  days. 
If  the  gelatin  in  any  of  the  tubes  becomes  cloudy,  the  medium 
in  those  tubes  must  be  rejected.  Carefully  wipe  all  the  other 
tubes  with  a  moist  cloth,  label,  and  place  them  in  the  locker, 
where  they  can  be  kept  until  used. 

18.  The  preparation  of  nutrient  agar.  Weigh  out  3  grams 
of  agar  and  cut  it  into  small  pieces  with  a  pair  of  scissors. 
Put  the  finely  cut  agar  into  an  agate-iron  dish,  add  75  cc. 
of  distilled  water,  and  boil  over  a  gas  flame,  with  constant 

1  For  illustrations  and  directions  for  folding  filter  paper,  see  Abbott's 
Principles  of  Bacteriology^  6th  edition,  p.  loi.  Filter  paper  already 
folded  may  be  procured. 


THE   PREPARATION  OF  GELATIN  AND  AGAR        1 3 

stirring  to  prevent  scorching,  until  the  agar  is  dissolved,  giv- 
ing a  thick,  homogeneous,  pasty  substance.  Pour  300  cc.  of 
bouillon  (§  11)  from  a  flask  into  the  cup  containing  the  dis- 
solved agar.  Place  the  dish  containing  the  mixed  agar  and 
bouillon  in  a  closed  water  bath  and  boil  for  20  minutes,  then 
cool  it  to  a  temperature  between  45°  and  50°  C,  add  the 
white  of  one  egg,  and  thoroughly  mix  in  the  liquid  agar. 
This  is  easily  accomplished  by  pouring  it  a  number  of  times 
from  one  beaker  to  another.  When  the  egg  albumen  is  dis- 
solved the  agar  is  returned  to  the  water  bath  and  boiled  vig- 
orously until  the  white  of  the  egg  is  firmly  coagulated.  This 
usually  takes  about  20  minutes.  Filter  the  agar  immediately, 
while  hot,  through  ordinary  filter  paper  which  has  been  mois- 
tened with  boiling  water.  Distribute  the  filtrate  in  small  and 
large  tubes,  as  directed.  Sterilize,  label,  and  store  the  agar  in 
the  same  manner  as  the  gelatin. 


14  LABORATORY   BACTERIOLOGY 


EXERCISE   V 

INOCULATING  TUBES   OF  BOUILLON,  AGAR,  AND 
GELATIN 

19.  Work  for  this  exercise.  See  that  the  media  made  in 
former  exercises  have  been  properly  sterihzed.  Inoculate  one 
tube  of  bouillon,  two  (one  inclined,  the  other  not)  of  agar,  and 
one  of  gelatin  from  a  culture  of  Bacillus  coli  communis^  which 
will  be  furnished. 

Wipe  the  slides.  Transfer  the  cover  glasses  from  the  clean- 
ing mixture  to  water  and  then  to  alcohol.  Seal  the  agar  slant 
and  gelatin  tubes. 

Read  the  chapters  in  one  or  more  text-books  on  inoculating 
media  or  making  tube  cultures. 

20.  Inoculating  bouillon.  In  making  this  culture,  carefully 
remove  the  plug  from  the  tube  of  bouillon  by  first  twisting  it 
around  to  detach  any  adhesions  and  then  by  pulling  it  straight 
out.  Pass  the  open  end  of  the  tilted  tube  quickly  through  the 
gas  flame.  The  plug,  which  has  meantime  been  carefully  held, 
is  partially  replaced  and  the  tube  returned  to  its  stand.  Treat 
the  tube  containing  the  culture  (which  has  been  furnished)  in 
the  same  manner.  Then  place  the  two  tubes  side  by  side 
between  the  thumb  and  forefinger  of  the  left  hand,  palm  facing 
upward,  and  grasp  them  about  the  middle  of  the  upper  half 
(see  Fig.  38,  p.  108,  Crookshank).  Sterilize  the  platinum 
loop  by  passing  it  through  the  gas  flame,  care  being  taken  that 
the  handle  is  also  flamed  for  a  distance  of  at  least  15  cm. 
Then  carefully  remove  the  plugs  from  the  tubes  and  hold  them 
between  the  fingers  in  such  a  manner  that  the  tube  ends,  pro- 
jecting outward,  will  not  touch  anything  during  the  inoculation 
process.  Insert  the  wire  loop  carefully  into  the  culture  and 
transfer  a  loopful  of  the  culture  to  the  tube  of  bouillon  and 
gently  rinse  it  from  the  loop.     The  loop  is  then  withdrawn,  the 


INOCULATING  TUBES   OF  BOUILLON  1 5 

plugs  replaced  in  their  respective  tubes,  and  the  loop  flamed 
and  put  aside.  Label  the  freshly  inoculated  tube  with  the 
name  of  the  organism,  source,  and  date.  Stand  it  in  a  tray  or 
cup  and  place  it  in  the  incubator.^  This  should  be  kept  at  a 
temperature  between  35°  and  37°  C.  The  organism  thus  trans- 
ferred should  multiply  so  that  on  the  following  day  the  liquid 
will  be  cloudy.  It  is  then  a  bouillon  culture  of  B,  coli  com- 
munis, 

21.  Inoculating  tubes  of  agar.  Ordinarily  the  agar  is 
i7iclined  before  it  is  i?ioculated.  In  this  case  it  is  spoken  of  as 
inclined  or  slant  agar.  Occasionally  the  agar  is  inoculated 
without  inclining  it.  Cultures  made  in  this  manner  are  spoken 
of  as  "stab"  or  "stick"  cultures,  {a)  Inclined  or  slant  agar. 
Stand  a  tube  of  agar  in  a  wire  basket  in  a  water  bath  and  boil 
it  until  the  agar  is  liquefied.  (To  save  repeating  this  it  is 
well  to  incline  the  agar  in  several  tubes  which  can  be  kept 
for  future  use,  but  after  the  slants  have  been  made  for  a  long 
time  it  is  better  to  boil  and  reslant  them,  especially  if  they 
are  to  be  used  for  organisms  which  do  not  grow  well  on  a  dry 
surface.)  Lay  the  tube  on  a  tray,  the  top  resting  on  the  side 
of  the  tray  so  that  the  surface  of  the  agar  will  be  about  4  cm. 
long,  and  allow  it  to  cool.  In  placing  the  tubes  the  label 
should  be  up.  When  the  agar  has  set  it  is  ready  for  use.  It  is 
inoculated  precisely  as  the  bouillon,  excepting  that  the  loop- 
ful  of  culture  is  drawn  over  the  inclined  surface  instead  of 
being  thrust  into  the  medium  as  in  the  bouillon.  Label  and 
place  it  in  the  incubator  with  the  inoculated  bouillon  tube. 
On  the  following  day  there  should  be  a  grayish-white  growth 

1  For  illustrations  and  descriptions  of  different  kinds  of  incubators, 
see  text-books.  It  is  desirable  to  note  especially  the  various  burners  and 
thermoregulators  employed  to  heat  and  regulate  the  temperature  of 
the  incubators.  Considerable  information  may  also  be  acquired  by 
carefully  looking  through  the  catalogues  of  manufacturers  and  dealers 
in  bacteriologic  apparatus.  Copies  of  some  of  these  will  be  found  on 
the  reference  bookshelves. 


1 6  LABORATORY   BACTERIOLOGY 

on  the  surface  of  the  agar  covered  by  the  loop.  This  is  an 
agar  culture  of  B,  coli  communis,  {b)  Stick  cultures.  These 
are  made  with  a  platinum  needle  in  the  uninclined  agar.  The 
impregnated  needle  is  pushed  down  through  the  center  of  the 
agar.  In  all  other  respects  this  culture  is  made  like  the  slant- 
agar  culture. 

22.  Inoculating  tubes  of  gelatin.  Tube  cultures  in  gela- 
tin are  usually  made  without  incHning  the  gelatin,  i.e.  stick 
cultures.  The  tube  of  gelatin  is  inoculated  in  the  same  man- 
ner as  the  stick  culture  in  agar.  This  tube  is  to  be  placed  in 
the  locker,  as  the  gelatin  will  melt  at  the  incubator  tempera- 
ture. The  growth  will  appear  along  the  needle  track  in  about 
two  days.     This  is  a  gelatin  culture  of  B.  coli  communis, 

23.  Sealing  culture  tubes.  It  is  often  desirable  to  seal 
cultures  to  prevent  their  drying  out  quickly.  A  convenient 
method,  and  one  which  has  long  been  in  use  in  some  labora- 
tories, is  to  boil  a  small  quantity  of  paraffin  in  a  small  agate- 
iron  dish  and  while  it  is  still  hot  carefully  dip  the  tube  end 
of  the  plug  into  it  and  quickly  replace  it  in  the  tube.  The 
paraffin  on  cooling  fills  the  spaces  between  the  fibers  of  cotton 
and  also  adheres  to  the  sides  of  the  tube,  forming  a  tight  plug. 
When  the  tube  is  to  be  opened  the  end  must  be  warmed 
slightly  before  the  plug  can  be  withdrawn.  The  plugs  should 
be  paraffined  and  the  sterility  of  the  tubes  determined  before 
they  are  used  for  cultures. 


THE  EXAMINATION   OF  CULTURES  1/ 

EXERCISE   VI 

THE   EXAMINATION   OF   CULTURES 

24.  In  studying  cultures  of  bacteria  it  is  necessary  to 
observe  very  carefully  (i)  the  macroscopic  appearance  of  the 
growth  in  or  upon  the  media,  (2)  the  microscopic  appearance 
of  the  bacteria  in  (a)  the  living  condition  (hanging-drop  prep- 
aration) and  (l^)  in  the  dead  and  stained  condition  (cover- 
glass  preparation),  and  (3)  the  effect  of  the  growth  of  the 
bacteria  upon  the  chemical  and  physical  properties  of  the 
medium.  To  determine  these  the  cultures  must  be  kept 
under  observation  for  several  days  and  often  for  several  weeks. 
A  careful  record  should  be  made  of  the  changes  observed  in 
the  appearance  of  the  cultures.     Illustrate  with  drawings. 

25.  Work  for  this  exercise.  Examine  carefully  and  de- 
scribe fully  the  appearance  of  the  bouillon,  agar,  and  gelatin 
cultures  made  in  Exercise  V. 

Determine  the  reaction  of  the  bouillon  culture  and  note 
whether  there  is  any  change  in  its  consistence  (viscidity). 

Make  a  hanging-drop  preparation  from  each  culture  and 
examine  and  describe  the  appearance  of  the  bacteria  in 
each. 

Make  a  drawing  of  the  gelatin  and  slant-agar  cultures  and 
also  of  a  few  of  the  bacteria  in  one  of  the  hanging-drop 
preparations. 

Read  the  paragraphs  in  one  or  more  text-books  on  the 
examination  of  cultures  and  hanging-drop  preparations. 

26.  Suggestions  for  the  macroscopic  examination  of  cultures. 
The  external  appearance  of  cultures  should  be  observed  and 
noted  on  the  day  after  they  are  made  and  on  each  succeeding 
day  until  the  growth  ceases.  In  bouillon  cultures  note  the 
appearance  of  the  liquid,  whether  uniformly,  faintly,  or  heavily 
clouded,  turbid,  clear,  or  clouded  with  flocculent  masses  held 


I  8  LABORATORY  BACTERIOLOGY 

in  suspension,  the  quantity  and  nature  of  sediment,  and  the 
presence  or  absence  of  a  membrane.  The  reaction  of  the 
liquid  should  be  taken  and  its  consistence  noted.  The  odor 
should  be  determined.  In  agar  cultures  the  extent  of  the 
growth  (feeble,  moderate,  or  vigorous),  its  color,  form,  and 
surface  appearance  (dull  or  glistening),  should  be  observed. 
The  character  of  the  growth  in  the  condensation  water  should 
also  be  noted.  In  stab  cultures  the  appearance  of  the  growth 
both  on  the  surface  and  along  the  needle  track  should  be 
described.  In  gelatin  the  absence  or  the  presence  and  extent 
of  liquefaction  should  be  noted  in  addition  to  the  features 
already  referred  to  for  the  stab-agar  cultures.  (See  Chester's 
terminology,  §§31  and  51.) 

27.  Testing  the  reaction  of  liquid  cultures.  Place  a  small 
piece  of  each  of  the  red  and  blue  Htmus  papers  in  a  solid 
watch  glass.  With  the  platinum  loop  carefully  place  a  drop 
of  the  culture  on  each  piece  of  the  paper.  After  recording 
the  reaction  produced,  —  neutral,  acid,  or  alkaline,  with  the 
degree,  —  cover  the  paper  with  a  disinfectant  (a  solution  of 
corrosive  sublimate  i  to  looo).  After  it  has  acted  for  about 
lo  minutes,  empty  it  with  the  paper  into  the  waste  jar  and 
wash  the  watch  glass. 

28.  To  determine  the  viscidity,  (a)  Bouillon  cultures. 
Insert  the  platinum  loop  into  the  liquid  and  carefully  with- 
draw it.  The  approximate  degree  of  viscidity  may  be  deter- 
mined by  the  extent  of  the  adhesion  of  the  liquid  to  the  loop 
and  by  the  length  of  the  threadlike  filament  drawn  out.  By 
gently  shaking  the  tube,  a  viscid  sediment  will  rise  up,  appear- 
ing as  a  somewhat  twisted,  tenacious  cone  with  its  apex 
reaching  to  or  near  the  surface.  A  friable  sediment  will  break 
up  and  become  disseminated  through  the  liquid  upon  agita- 
tion. (/;)  Agar  and  gelatin  cultures.  Touch  the  surface 
growth  with  the  end  of  the  platinum  needle,  and  if  it  is  viscid, 
a  threadlike  string  will  be  drawn  out.  Note  whether  the 
growth  is  pasty  or  friable. 


THE   EXAMINATION   OF  CULTURES  I9 

29.  Making  hanging-drop  preparations,  (a)  Fro7n  a  bou- 
illon culture.  Place  a  clean  cover  glass  on  a  tray.  With  the 
loop  remove  a  drop  of  the  liquid  culture  and  place  it  on  the 
middle  of  the  cover  glass.  With  a  pair  of  fine  forceps  invert 
the  cover  glass  over  the  glass  ring  fixed  to  a  slide  for  this 
purpose.  The  surface  of  the  ring  should  previously  be  mois- 
tened with  liquid  vaseline  to  prevent  the  cover  glass  from  slid- 
ing. The  preparation  is  then  ready  for  examination.  Examine 
it  first  with  the  high-power  dry  lens  and  then  with  the  oil- 
immersion  objective.  (For  directions  on  the  use  of  the 
microscope,  see  The  Microscope^  by  Professor  S.  H.  Gage.) 
{h)  From  cultures  on  solid  media.  On  account  of  the  very 
large  number  of  bacteria  in  the  growth  on  solid  media  it  is 
necessary  to  separate  them  in  a  clear  liquid.  Take  a  cover 
glass  as  before  and  place  a  loopful  of  bouillon  or  sterilized 
water  on  the  center.  Touch  the  surface  growth  very  gently 
with  the  end  of  the  platinum  needle  and  carefully  rinse  it  in 
the  drop  of  liquid  on  the  cover  glass.  From  this  point  the 
examination  is  the  same  as  with  the  liquid  culture.  Upon 
examination,  if  the  bacteria  are  so  numerous  that  the  indi- 
vidual organisms  cannot  be  clearly  distinguished,  i.e.  separated 
from  each  other,  the  preparation  must  be  rejected  and  another 
one  made,  using  a  smaller  quantity  of  the  growth.  After 
examination  the  cover  glasses  should  be  placed  at  once  in  a 
glass  jar  containing  a  strong  disinfectant  (5%  carbolic  acid, 
I  to  1000  corrosive  sublimate  solution,  or  a  strong  solution 
of  a  mineral  acid). 

30.  Suggestions  for  the  microscopic  examination  of  living 
bacteria.  In  examining  the  bacteria  as  they  appear  under , 
the  microscope  in  the  hanging-drop  preparation  the  following 
features  should  be  observed :  Are  the  individual  bacteria 
spherical,  rod-shaped,  or  spiral  in  form?  Are  they  single  or 
united  in  pairs,  masses,  or  clumps,  or  in  shorter  or  longer 
chains?  For  this  determination  it  is  better  to  examine  the 
organisms  near  the  edge  of  the  drop.    Are  they  motile,  that  is, 


20  LABORATORY   BACTERIOLOGY 

do  the  individual  bacteria  move  from  one  point  in  the  field 
to  another?  To  determine  this  the  center  of  the  drop  is 
better.  Clearly  distinguish  between  genuine  motility  and  a 
simple  dancing  motion  (the  Brownian  movement).  Deter- 
mine the  presence  or  absence  of  spores.  These  are  bright, 
highly  refractive  bodies  either  within  or  outside  the  bodies 
of  the  bacteria.  If  present,  they  can  usually  be  seen  in  both 
positions.  Is  there  any  evidence  of  a  capsule  around  the 
bacteria  ? 

31.  Chester*s  terminology  for  descriptive  bacteriology.  Ches- 
ter has  introduced  a  terminology  in  descriptive  bacteriology 
which  has  the  advantage  of  being  definite  and  concise,  while 
at  the  same  time  it  is  sufficiently  elastic  to  fit  the  varying 


Fig.  I.    Characters  of  surface  elevation:  i,  flat;  2,  raised;  3,  convex; 
4,  pulvinate  ;  5,  capitate  ;  6,  umbilicate  ;  7,  umbonate. 

forms  of  growth.  It  applies  to  the  surface  growth,  to  the  growth 
along  the  needle  track  in  the  depth  of  the  media,  and  to 
colonies  on  plate  cultures. 

I.  Surface  elevation.     General  character  of  surface  growth  as 
a  whole. 

J^/a^ :  thin,  leafy,  spreading  over  the  surface. 

Effused:  spread  over  the  surface  as  a  thin,  veilly  layer, 

more  delicate  than  the  preceding. 
Raised :  growth  thick,  with  abrupt  terraced  edges. 
Convex:  surface  the   segment  of  a  circle,  but  very  flatly 

convex. 
Pulvinate :  surface  the  segment  of  a  circle,  but  decidedly 

convex. 
Capitate :  surface  hemispherical. 


THE  EXAMINATION   OF  CULTURES 


21 


££-- 


Fig.  2.    Characters  of  growth  in  depth  of  media:  i,  filiform  ;  2,  beaded; 
3,  tuberculate-echinulate ;  4,  arborescent ;  5,  villous. 


^    ©    a    ® 


Crr 


KMm 


Fig.  3.    Types  of  liquefaction  in  gelatin  stab  cultures:   i,  craterif orm ; 
2,  napiform  ;  3,  saccate  ;  4,  infundibuliform ;  5,  stratiform. 


22  LABORATORY  BACTERIOLOGY 

2 .  Gelatin  stab  cultures.     Nonliquef ying  line  of  puncture. 

Filiform  :  uniform  growth,  without  special  character. 
Nodose :  consisting  of  closely  aggregated  colonies. 
Beaded:  consisting  of  loosely  placed  or  disjointed  colonies. 
Papillate  :  beset  with  papillate  extensions. 
Echinate  :  beset  with  acicular  extensions. 
]/illous  :  beset  with  short,  undivided,  hairlike  extensions. 
Plumose :  a  delicate  feathery  growth. 

Arborescent :  branched,  or  treelike,  beset  with  branched 
hairlike  extensions. 

3.  Gelatin  stab  culture.     Liquefying  line  of  puncture. 

Crateriform :  a  saucer-shaped  liquefaction  of  the  gelatin. 

Saccate :  shape  of  an  elongated  sack,  tubular,  cylindrical. 

Infundibuliform :  shape  of  a  funnel,  conical. 

Napiform  :  shape  of  a  turnip. 

Fusiform  :  outline  of  a  parsnip,  narrow  at  either  end,  broad- 
est below  the  surface. 

Stratiform :  liquefaction  extending  to  the  walls  of  the  tube 
and  downward  horizontally. 


MAKING  COVER-GLASS    PREPARATIONS  23 


EXERCISE   VII 

MAKING  AND  STAINING  COVER-GLASS  PREPARATIONS, 
AND   FORMULAE   FOR  STAINING   SOLUTIONS 

32.  Work  for  this  exercise.  Make  2  cover-glass  prepa- 
rations from  each  of  the  cultures  made  in  Exercise  V  and  stain 
one  of  each  with  alkaline  methylene  blue  and  the  other  with 
carbol  fuchsin.  Describe  the  appearance  of  the  bacteria  and 
make  a  drawing  of  a  few  individual  bacteria  from  the  prepara- 
tions made  from  the  agar  culture. 

Preserve  a  cover-glass  preparation  mounted  in  balsam  and 
labeled  to  accompany  notes. 

Prepare  the  staining  fluids  used  in  this  exercise  from  the 
formulae  given  (§37). 

Read  the  paragraphs  in  the  text-books  on  making  and  stain- 
ing cover-glass  preparations.  See  references  and  text-books 
for  various  staining  solutions  that  are  sometimes  used. 

33.  Making  cover-glass  preparations,  (a)  From  houillo7i 
cultures.  Place  two  clean  cover  glasses  on  the  tray.  With  the 
loop  remove  a  drop  of  the  bouillon  culture  and  spread  it  in  a 
thin  layer  over  about  two  thirds  of  the  surface  of  the  cover 
glasses.  One  loopful  will  ordinarily  make  from  2  to  4  prepara- 
tions. Allow  the  liquid  to  dry  on  the  cover  glasses  in  the 
•air  and,  when  dry,  fix  the  bacteria  to  them  by  passing 
them,  film  upward,  three  times  through  the  middle  of  the 
upper  half  of  the  gas  flame.  Each  passage  (complete  circle) 
should  not  occupy  more  than  one  second.  After  fixing  they  are 
ready  for  staining,  {b^  From  cultures  on  solid  media  {agar, 
gelatin,  potato,  serum,  etc.).  Place  the  cover  glasses  on  the 
tray,  and  on  the  center  of  each  put  a  drop  of  sterile  water  or 
bouillon.  Touch  the  surface  growth  of  the  culture  with  the 
end  of  the  needle  and  then  gently  rinse  it  in  the  liquid  on 
the  covers.    Spread  the  liquid  on  the  covers  as  before.     From 


24  LABORATORY  BACTERIOLOGY 

this  point  the  procedure  is  the  same  as  that  for  the  preparations 
made  from  the  bouillon  culture. 

34.  Staining  bacteria  in  cover-glass  preparations,  (a)  With 
alkaline  methylene  blue.  With  the  pipette  place  a  few  drops 
of  the  staining  solution  on  the  film  side  of  the  fixed  prepa- 
ration, which  is  either  held  horizontally  with  the  fine  forceps 
or  left  resting  on  the  tray.  Allow  the  stain  to  act  for  2  or 
3  minutes;  then  carefully  rinse  it  off  in  water,  holding  the 
cover  firmly  by  one  edge  with  the  forceps.  After  thoroughly 
rinsing,  place  the  preparation,  film  downward,  on  a  clean  slide 
and  dry  the  upper  surface  with  a  piece  of  filter  paper.  It  is 
now  ready  for  the  microscopic  examination.  Use  first  the 
dry  lens  (J-in.  obj.)  and  then  the  oil-immersion  objective.  If 
the  specimen  is  a  good  one  and  it  is  desirable  to  preserve  it, 
wipe  off  the  drop  of  oil  with  a  piece  of  lens  paper  and  run  a 
drop  of  distilled  water  under  the  cover  glass,  which  will  float  it, 
when  it  can  be  easily  removed  with  the  forceps.  Place  it  on 
the  tray,  film  upward,  and  when  dry,  mount  it  in  a  neutral  or 
slightly  alkaline  Canada  balsam.^ 

{h)  With  carhol  fuchsin.  Moisten  the  film  side  of  the 
cover  glass  with  water  (using  the  pipette);  then  cover  it  with 
the  stain  and  allow  it  to  act  for  from  10  to  30  seconds.  Then 
rinse  it  thoroughly  in  water,  after  which  cover  it  with  xV% 
solution  of  acetic  acid  or  strong  (95%)  alcohol.  Allow  this 
to  act  for  from  5  to  10  seconds,  and  again  thoroughly  rinse 
in  water  and  examine  as  above.  (For  other  decolorizers,  see 
text-books.) 

Upon  examination  the  preparation  should  be  free  from 
deposits  or  stained  background.  The  bacteria  should,  as  a 
rule,  be  isolated  and  distinct ;  unless  they  are  the  preparations 
are  not  satisfactory. 

^  To  neutralize  balsam,  add  some  pure  sodium  carbonate  to  it  and 
allow  it  to  stand  for  about  a  month  in  a  warm  place,  shaking  it  from 
time  to  time.  Then  allow  the  sodium  to  settle.  The  clear  supernatant 
balsam  will  be  found  to  be  slightly  alkaline. 


MAKING  COVER-GLASS   PREPARATIONS  2$ 

Cover-glass  preparations  of  bacteria  are  permanently  mounted 
in  the  same  manner  as  similar  preparations  made  from  the 
blood  or  other  tissues  in  histology,  the  process  being  to  put  a 
drop  of  balsam  on  the  center  of  the  slide  and  place  the  prep- 
aration, film  downward,  over  it  and  apply  slight  pressure. 
Label  the  preparation,  giving  the  name  of  the  organism,  its 
source  (kind  of  culture,  tissue,  etc.,  from  which  the  preparation 
was  made),  stain  used,  and  date.  If  the  specimen  is  not  pre- 
served, the  slide  and  cover  glass  should  be  cleaned  for  future  use. 

35.  Suggestions  concerning  the  microscopic  examination  of 
stained  preparations  of  bacteria.  In  the  examination  of  the 
bacteria  in  the  stained  condition  the  following  points,  and 
perhaps  others,  should  be  observed  and  noted,  (a)  Concern- 
ing  their  morphology.  Are  they  spherical,  rod-shaped,  or 
spiral  ?  Are  they  separated  or  united  in  clumps  or  chains  ?  If 
rod-shaped,  are  the  ends  pointed,  round,  or  square?  Are  the 
bacteria  all  of  the  same  form  and  size?  Note  the  presence 
or  absence  of  spores,  granules,  and  capsules,  (h)  Coficerning 
their  reaction  to  stai7iing  fluids.  Do  they  stain  uniformly  or 
irregularly?  Do  they  stain  deeply  or  faintly?  Is  the  center 
lighter  than  the  periphery?  Are  there  an  unstained  central 
band  and  deeply  stained  ends  (polar  stain)?  Do  all  of  the 
bacteria  take  the  stain  alike? 

36.  Staining  solutions.  The  basic  aniline  dyes  are  used  in 
staining  bacteria.  There  is  a  large  number  of  these,  and  there 
are  several  formulae  for  preparing  staining  solutions  from 
each.  Further,  as  will  be  seen  from  the  chapters  on  staining 
bacteria  in  the  text-books,  there  are  several  methods  of  apply- 
ing these  stains.  In  an  introductory  course,  however,  it  is 
impossible  to  try  them  all,  and  consequently  only  those  are 
described  which  seem  to  be  the  best  adapted  for  general  use. 

In  addition  to  the  ordinary  staining  solutions  and  methods 
there  are  special  processes  for  certain  species,  such,  for  exam- 
ple, as  the  tubercle  bacterium,  and  still  others  for  staining 
certain  parts  of  many  bacteria,  such  as  the  flagella  on  motile 


26  LABORATORY  BACTERIOLOGY 

forms,  the  spores  in  spore-bearing  organisms,  and  the  capsule 
on  certain  other  species.  There  is  a  large  number  of  these 
special  methods,  but  in  this  course  only  one  of  each  will  be 
given.  These  will  be  taken  up  in  connection  with  the  study 
of  the  bacteria  requiring  them. 

37.  FormulaB  for  staining  solutions.  The  dyes  here  used 
are  methylene  blue,  gentian  violet,  methyl  violet,  and  basic 
fuchsin.     For  the  other  dyes,  see  text-books. 

ALKALINE    METHYLENE   BLUE    (LOEFFLER) 

Saturated  alcoholic  solution  of  methylene  blue  .     6     cc. 

Caustic  potash  (i  %  solution) 0.2  cc. 

Distilled  water 20     cc. 

The  saturated  alcoholic  solution  of  the  methylene  blue  (or 
of  any  of  the  dyes)  is  prepared  by  pouring  the  dye  into  a  clean 
bottle  and  filling  it  about  one  fourth  full.  Then  fill  the  bottle 
with  strong  (95  %  or  absolute)  alcohol,  cork  tightly,  shake,  and 
allow  it  to  stand  for  24  hours.  If  at  the  end  of  that  time  the 
dye  is  entirely  dissolved,  add  more,  shake  thoroughly,  and  allow 
it  to  stand  for  another  day.  Repeat  this  procedure  until  there 
is  a  permanent  sediment  of  undissolved  coloring  matter  in  the 
bottom  of  the  bottle.  Then  label.  (The  saturated  solution 
will  be  kept  in  stock  in  the  laboratory.) 

CARBOL  FUCHSIN  (ZIEHL'S  SOLUTION) 

Fuchsin  (dry) i  gram 

Alcohol  (absolute) 10  cc. 

Carbolic  acid  (5  %  solution) 100  cc. 

Dissolve  the  fuchsin  in  the  alcohol,  after  which  add  the  car- 
bolic acid  solution.  Instead  of  using  the  dry  fuchsin  and 
alcohol,  1 1  cc.  of  a  saturated  alcoholic  solution  of  fuchsin  may 
be  used. 

It  is  more  convenient  for  each  student  to  prepare  the 
following  :  — 

Saturated  alcoholic  solution  of  fuchsin  ....       3  cc. 
Carbolic  acid  (5%  solution) 20  cc. 


MAKING  COVER-GLASS  PREPARATIONS  27 

If  the  mixture  is  not  clear,  add  more  of  the  saturated  alco- 
holic solution  of  fuchsin  drop  by  drop  until  when  viewed 
through  the  pipette  by  transmitted  light  the  hquid  is  perfectly 
clear. 

ANILINE    GENTIAN    VIOLET    (EHRLICH-WEIGERT) 

Saturated  alcoholic  solution  of  gentian  violet      .     11  cc. 

Absolute  alcohol 10  cc. 

Aniline  water 100  cc. 

It  is  more  convenient  for  each  student  to  prepare  one  fifth 
of  this  quantity. 

CARBOLIC   THIONINE   BLUE    (NICOLLE) 

Thionine  blue i     gram 

CarboHc  acid 2.5  grams 

Distilled  water loo     cc. 

Filter.  Before  using,  dilute  with  an  equal  quantity  of  distilled 
water  and  filter  again. 

CARBOLIC    GENTIAN    VIOLET    (NICOLL^) 

Gentian  violet  (saturated  alcoholic  solution)       .     10  cc. 
Carbolic  acid  (i  %  solution) 100  cc. 

Mix  and  filter  before  storing. 

38.  Aqueous  solutions.  Aqueous  solutions  of  methyl  violet, 
gentian  violet,  fuchsin,  and  the  other  aniline  dyes  are  prepared 
by  adding  i  cc.  of  the  saturated  alcoholic  solution  of  the 
desired  dye  to  20  cc.  of  distilled  water.  This  will  impart  a 
decided  color  to  the  liquid,  so  that  in  a  pipette  it  will  be  barely 
transparent. 

The  true  aqueous  solutions  are  made  by  dissolving  the  dyes 
in  water,  but  these  are  weak  and  not  so  effective  as  those  pre- 
pared from  the  alcoholic  solutions.  These  solutions  deteriorate 
in  a  short  time.  The  carbol  fuchsin  and  alkaline  methylene 
blue  will  keep  a  little  longer,  but  they  require  filtering  occa- 
sionally. 


28  LABORATORY  BACTERIOLOGY 

39.  Making  aniline  water.  Aniline  water  is  a  saturated 
aqueous  solution  of  aniline  oil.  It  is  prepared  by  adding  i  cc. 
of  aniline  oil  to  20  cc.  of  distilled  water  and  shaking  frequently 
for  from  15  to  30  minutes.  It  is  convenient  to  use  a  stoppered 
vial  or  large  test  tube  for  mixing  it.  Filter  through  a  mois- 
tened filter  paper.  The  filtrate  should  be  perfectly  clear.  If 
it  is  cloudy,  it  should  be  refiltered  before  using.  This  is  used 
in  preparing  the  aniline  water  dyes,  such  as  methyl  violet, 
gentian  violet,  etc. 

40.  Gram*s  method  of  staining  bacteria.  Prepare  the  cover- 
glass  preparations  as  already  described.  Stain  them  in  gentian- 
violet  aniline  water,  or  in  a  saturated  alcohoHc  solution  of 
gentian  violet  in  5  %  carbolic  acid  in  the  proportion  of  i  to  20 
for  from  5  to  7  minutes.  Rinse  in  water  and  transfer  them  to 
a  watch  glass  containing  Gram's  solution  until  the  color  becomes 
quite  black.  This  requires  from  i  to  2  minutes;  then  place 
the  preparations  in  a  watch  glass  containing  alcohol  and  allow 
them  to  remain  there  until  the  color  has  almost  entirely  dis- 
appeared, or  has  become  a  pale  gray.  Rinse  in  water  and 
examine  at  once,  or  allow  them  to  dry  and  mount  in  balsam. 
(Sections  of  tissues  must  be  dehydrated  and  cleared  before 
mounting.) 

gram's  solution  (lugol's) 

Iodine i  gram 

Potassium  iodide 2  grams 

Distilled  water •     •     •  S^o  cc. 

Certain  bacteria  stain  deeply  and  retain  the  coloring  matter 
when  treated  by  this  method,  while  others  are  decolorized  by 
the  alcohol.  On  this  account  some  investigators  consider  it  an 
important  aid  in  the  differentiation  of  certain  bacteria. 


MAKING  PLATE  AND  ESMARCH  ROLL  CULTURES      29 

EXERCISE   VIII 

MAKING  PLATE   AND   ESMARCH  ROLL  CULTURES 

41.  The  general  principle  underlying  the  separation  of  bac- 
teria by  means  of  plate  and  roll  cultures  is  to  dilute  the  sub- 
stance containing  the  bacteria  so  that  the  individual  organisms 
will  be  separated  from  each  other  by  an  appreciable  distance 
and  then  fixed  in  a  solid  medium  where  each  organism  can 
multiply  into  a  growth  or  colony  without  coming  in  contact 
with  any  other  organism  or  colony.  For  this  purpose  agar 
and  gelatin  are  used.  Originally  Koch  employed  a  rectangu- 
lar piece  of  glass  for  holding  the  layer  of  medium,  and  pro- 
tected it  from  contamination  by  putting  it  under  a  bell  jar. 
Later  Esmarch  introduced  the  "roll-culture"  method,  which 
was  extensively  followed  until  the  Petri  dishes  were  intro- 
duced. Since  that  time  the  latter  have  been  largely  used  in 
place  of  the  Koch  plate  and  Esmarch  tube.  On  this  account 
the  plate  cultures  of  to-day  are  usually  made  in  Petri  dishes. 
The  roll  culture  is  also  used. 

Plate  cultures  are  employed  for  two  distinct  purposes : 
(i)  to  isolate  bacteria  in  order  to  obtain  pure  cultures  from 
the  isolated  colonies ;  and  (2)  to  determine  how  many  bacteria 
there  are  present  in  a  given  quantity  of  a  liquid  such  as  water, 
milk,  or  blood.  In  this  exercise  the  object  is  to  separate  the 
bacteria  to  obtain  isolated  colonies.  For  quantitative  work, 
see  Exercise  LV. 

42.  Work  for  this  exercise.  Make  a  series  of  3  agar  plates, 
one  of  3  gelatin  plates,  and  one  of  3  gelatin  roll  cultures 
(Esmarch  rolls)  from  the  bouillon  culture  of  B,  colt  communis 
(§  19).  Place  the  agar  plates  in  the  incubator  and  the  gelatin 
plates  and  rolls  in  a  locker  for  that  purpose. 

Reexamine  all  the  cultures  made  in  previous  exercises  and 
add  to  the  laboratory  notes  a  description  of  any  changes  in 


30  LABORATORY  BACTERIOLOGY 

their  appearance.     The  notes  should  contain  a  detailed  record 
of  the  cultures  made  in  this  exercise. 

Read  carefully  the  paragraphs  in  the  text-books  on  making 
plate  and  roll  cultures. 

43.  Making  agar  plate  cultures.  Take  3  large  tubes  of 
agar,  stand  them  in  a  water  bath,  and  boil  until  the  agar  is 
liquefied.  Then  cool  by  standing  the  tubes  with  a  thermome- 
ter in  a  cup  of  water  at  a  temperature  of  about  50°  C.  As  the 
temperature  rises,  add  a  little  cold  water.  When  the  tempera- 
ture of  the  agar  reaches  that  of  the  water,  and  the  temperature 
cf  the  whole  has  lowered  to  40°  C,  the  agar  is  ready  for  use. 
For  convenience  in  labeling,  number  the  tubes  1,2,  and  3. 

Place  3  sterilized  Petri  dishes  on  the  leveling  tripod  and 
adjust  it  by  means  of  a  spirit  level.  With  the  wire  loop  pro- 
ceed by  the  same  method  as  followed  in  making  bouillon 
cultures.  Take  one  loopful  of  the  bouillon  culture,  place  it 
in  agar  tube  i  and  mix  by  carefully  shaking.  Flame  the 
wire  and  transfer  2  loopfuls  of  agar  from  tube  i  to  tube  2 
and  mix  as  before.  Again  flame  the  loop  and  transfer  3  loop- 
fuls from  tube  2  to  tube  3  and  mix  as  with  tubes  i  and  2. 
After  the  tubes  are  inoculated,  pour  the  agar  into  the  Petri 
dishes.  In  doing  this  remove  the  plug,  carefully  flame  the 
mouth  of  the  tube,  and  after  quickly  cooling,  raise  with  the 
left  hand  the  edge  of  the  cover  on  one  side  of  the  Petri  dish 
sufficiently  to  allow  of  inserting  the  mouth  of  the  tube.  After 
the  agar  is  poured  out  of  the  tube  replace  the  cover  immedi- 
ately. Label  and  number  the  Petri  dishes  to  correspond  with 
the  dilutions  in  the  tubes  :  thus,  plate  i  is  from  tube  i ,  plate  2 
is  from  tube  2,  and  plate  3  is  from  tube  3.  Place  the  label 
near  the  edge  of  the  cover.  The  Faber  pencil  for  marking 
glass  may  be  used  instead  of  the  gummed  label.  In  making 
the  dilutions  it  is  important  that  the  wire  loop  should  be 
flamed  after  making  each  transfer. 

44.  Making  gelatin  plate  cultures.  These  are  prepared 
precisely  as  the  agar  plates,  with  these  exceptions  :   ( i )  the 


MAKING  PLATE  AND  ESMARCH  ROLL  CULTURES      3  I 

gelatin  is  liquefied  at  a  temperature  of  45°  C ;  (2)  the  plates 
when  made  are  to  be  kept  in  the  locker  the  same  as  the  gelatin 
stab  cultures;  (3)  in  hot  weather  it  is  sometimes  necessary 
to  put  a  piece  of  ice  in  the  reservoir  under  the  glass  plate  on 
the  leveHng  tripod  to  congeal  the  gelatin. 

The  directions  given  above  for  making  the  dilutions  are 
applicable  only  when  the  original  culture  is  moderately  clouded. 
If  there  are  comparatively  few  bacteria  in  the  liquid,  a  larger 
quantity  of  the  culture  will  be  necessary.  If  there  are  many 
more,  as  in  turbid  bouillon  or  in  slant- agar-culture  cultures, 
it  will  be  necessary  to  take  a  much  smaller  quantity  for  the 
first  dilution.  It  is  often  desirable  to  make  the  first  dilution 
in  a  tube  of  sterile  water  or  bouillon  instead  of  gelatin  or  agar, 
and  to  make  2  rather  than  3  plates.  It  is  sometimes  desirable 
to  make  4  or  more  cultures. 

45.  Making  Esmarch  roll  cultures.  For  this  purpose  gela- 
tin is  ordinarily  used.  Agar  does  not  adhere  readily  to  the 
sides  of  the  tubes,  but  is  sometimes  used.  Take  the  desired 
number  of  large  tubes  of  gelatin,  liquefy,  inoculate,  label,  and 
number  the  dilutions  as  in  making  gelatin  plate  cultures. 
Place  a  block  of  ice  about  6  inches  long  in  an  agate-iron  or 
glass  tray.  Melt  a  slight,  nearly  horizontal  groove  in  the  ice 
with  a  test  tube  containing  hot  media  or  water.  The  inocu- 
lated tubes  are  tipped  and  rolled  so  that  the  liquid  gelatin 
moistens  the  inside  of  the  tube  to  within  about  a  centimeter 
of  the  plug.  Then  roll  the  tube  rapidly  in  the  groove  on  the 
ice  until  the  medium  becomes  solid.  The  gelatin  should  not 
come  in  contact  with  the  plug.  In  rolling  the  tube  the  plugged 
end  should  always  project  beyond  the  ice.  (See  illustration  in 
text-books.) 


32  LABORATORY  BACTERIOLOGY 


EXERCISE    IX 

THE  EXAMINATION   OF  PLATE   CULTURES   AND  THE 
MAKING  OF  SUBCULTURES   FROM  COLONIES 

46.  In  practical  bacteriologic  work  plate  cultures  are  made 
use  of  in  determining  (i)  the  number  of  bacteria  there  is  in 
a  given  substance,  (2)  the  different  species  of  bacteria  pres- 
ent, and  (3)  the  character  of  the  growth  in  a  colony  of 
the  organism  in  question.  Other  important  facts,  such,  for 
example,  as  the  relative  number  of  each  species  of  bacteria 
or  the  difference  in  the  appearance  of  the  surface  and  deep 
colonies,  are  learned  through  this  process.  The  plate  cul- 
ture, therefore,  is  one  of  the  most  important  single  methods 
employed  in  isolating  and  studying  bacteria. 

47.  Work  for  this  exercise.  Examine  carefully  and  describe 
the  plate  cultures  made  in  Exercise  VIII.  If  the  agar  plates 
do  not  have  colonies,  or  if  the  colonies  are  so  numerous  that 
they  cannot  be  counted  on  any  of  the  plates,  make  the  cultures 
over  again,  and  give  an  explanation  in  the  notes  of  this  exer- 
cise for  the  failure  to  obtain  good  results. 

Make  a  hanging-drop  preparation  from  a  colony  from  an 
agar  plate  and  one  from  a  colony  from  a  gelatin  plate,  and 
examine  them  microscopically.  Describe  the  appearance  of 
the  bacteria  in  each. 

Make  a  cover-glass  preparation  from  each  of  the  same 
colonies  and  stain  each  with  carbol  fuchsin.  Examine  each 
preparation  carefully  and  make  a  drawing  of  a  few  of  the 
isolated  bacteria.  Describe  (§  35)  the  appearance  of  the  bac- 
teria in  these  preparations. 

Inoculate  a  tube  of  bouillon  and  one  of  agar  from  a  well- 
isolated  colony  on  one  of  the  agar  plates. 

48.  Suggestions  for  the  examination  of  the  plate  and  rftll 
cultures.    Observe  the  general  appearance  of  the  plates;  note 


THE  EXAMINATION  OF   PLATE  CULTURES  33 

whether  the  colonies  are  well  isolated  or  run  together  (con- 
fluent) ;  describe  the  appearance  of  the  individual  colonies 
(a)  on  the  surface,  (d)  in  the  depth  of  the  medium. 

Indicate  their  shape  (see  §  51).  Are  the  edges  sharply 
defined  ?  Is  the  margin  even  or  iri;egular  ?  Give  their 
size  (diameter  in  millimeters)  and  indicate  their  color  (deter- 
mine shade  from  a  color  chart  ^)  and  consistence.  Do  the 
surface  colonies  adhere  to  the  medium  or  can  they  be  easily 
removed?  Examine  them  with  a  low-power  lens  and  describe 
the  surface  markings,  if  any.  Also  indicate  the  difference 
in  color  as  observed  with  the  unaided  eye  and  with  the 
microscope. 

49.  Estimating  the  number  of  colonies  on  plates.  If  the 
number  of  colonies  is  not  large  (not  exceeding  100),  they  may 
all  be  counted  and  the  exact  number  recorded.  This  may  be 
done  with  the  third  plate.  When  the  number  is  larger  it  is 
more  convenient  to  divide  the  total  area  into  smaller  areas  and 
count  the  number  of  colonies  in  each  of  several  (20  to  40) 
of  the  small  areas.  Add  these  together  and  divide  the  sum  by 
the  number  of  areas  counted ;  the  quotient  gives  the  average 
number  on  one  area.  Multiply  this  quotient  by  the  number 
of  areas  containing  colonies,  and  the  product  will  be  the  num- 
ber of  colonies  on  the  plate.  This  latter  process,  however, 
gives  the  approximate  number  only. 

For  dividing  the  area  of  the  plate  into  smaller,  equal  areas, 
it  is  convenient  to  use  Wolffhugel's  counting  apparatus.  This 
was  devised  more  particularly  for  square  or  oblong  plates 
(Koch).  In  counting  the  colonies  on  the  Petri  dishes  Parkes'  ^ 
scheme  modified  by  Jeifers  ^  is  more  suitable.  It  consists  of 
a  disk  about  20  cm.  in  diameter,  divided  into  areas  of  a  square 
centimeter  each.  Place  the  Petri  dish  over  the  disk,  taking 
care  that  it  is  centered. 

^  Saccardo,  Chromotaxia  seu  Nomenclator  Colorum. 

2  VdixV.^^,  Journal  of  Pathology  and  Bacteriology ^  Vol.  IV,  p.  173. 

^  ]q^qxs,  Journal  of  Applied  Microscopy  ^^\.  I,  No.  3,  1898. 


34  LABORATORY   BACTERIOLOGY 

Count  the  number  of  colonies  in  several  (lo  to  40)  of  the 
areas  and  multiply  the  mean  number  by  the  number  of  areas 
covered.  This  product  gives  the  approximate  number  of 
colonies  on  the  plate. 

50.  Making  subcultures  from  colonies.  Select  the  tubes 
of  media  to  be  used  and  flame  the  mouths  as  heretofore 
described.  Select  a  colony  as  well  isolated  from  all  others  as 
possible.  With  the  left  hand  carefully  raise  the  edge  of  one 
side  of  the  cover  of  the  Petri  dish  and,  while  holding  it,  touch 
the  colony  with  the  needle,  replace  the  cover,  take  up  the  tube 
of  medium  and  inoculate  it.  If  bouillon  is  used  first,  a  tube 
of  agar  or  gelatin  can  be  inoculated  immediately  afterward 
without  recharging  the  needle.  If  more  cultures  are  to  be 
made,  it  is  necessary  to  charge  the  needle  again  from  the 
colony.  If  the  plate  is  to  be  rejected,  the  cover  may  be  entirely 
removed  in  the  beginning.  The  newly  inoculated  tubes  or 
subcultures  should  be  labeled  and~  treated  according  to  the 
directions  heretofore  given  for  handling  cultures.  These 
inoculated  tubes  should  be  pure  cultures.  It  sometimes 
happens,  however,  that  what  appears  to  be  a  single  colony 
consists  of  the  growths  of  two  organisms.  If  these  should  be 
of  different  species,  the  cultures  made  from  the  colony  would 
probably  be  impure.  These  impure  growths  (apparently 
single  colonies)  frequently  develop  on  plate  cultures  exposed 
to  the  air  for  some  time.  Single  particles  of  dust  often  carry 
two  or  more  bacteria. 

51.  Chester's  terminology  for  description  of  colonies. 

I.  Form  of  colonies.     Plate  culture. 

Punctifor7n  :  dimensions   too  slight  for  defining  form  by 

naked  eye,  minute,  raised,  semispherical. 
Round:  of  a  more  or  less  circular  outline. 
Irregular. 
Elliptical. 

Fusiform :  spindle-shaped,  tapering  at  each  end. 
Cochleate  :  spiral  or  twisted  like  a  snail  shell. 


THE  EXAMINATION   OF  PLATE  CULTURES  35 

Ajnceboid:  very  irregular,  streaming. 

Mycelioid :  a  filamentous  colony  with  the  radiate  character 

of  a  mold. 
Fila7nentous  :  an  irregular  mass  of  loosely  woven  filaments. 
Floccose :  of  a  densely  woolly  structure. 
Rhizoid :  of  an  irregular  branched,  rootiike  character,  as 

in  Bad.  7nycoides. 
Conglomerate  :  an  aggregate  of  colonies  of  similar  size  and 

form. 
Toruloid :  an  aggregate  of  colonies  like  the  budding  of  the 

yeast  plant. 
Rosulate  :  shaped  like  a  rosette. 

Detailed  character  of  surface. 

Sinooth :  surface  even,  without  any  of  the  following  dis- 
tinctive characters. 

Alveolate :  marked  by  depressions  separated  by  thin  walls, 
so  as  to  resemble  a  honeycomb. 

Punctate  :  dotted  with  punctures  like  pin  pricks. 

Bullate :  like  a  blistered  surface,  rising  in  convex  promi- 
nences, rather  coarse. 

Vesicular:  more  or  less  covered  with  minute  vesicles,  due  to 
gas  formation  ;  more  minute  than  bullate. 

Verrucosa :  wartlike,  bearing  wartlike  prominences. 

Squamose :  scaly,  covered  with  scales. 

Echinate  :  beset  with  pointed  prominences. 

Papillate :  beset  with  nipple-  or  mamma-like  processes. 

Rugose:  short,  irregular  folds,  due  to  shrinkage  of  surface 
growth. 

Corrugated :  in  long  folds,  due  to  shrinkage. 

Contoured:  an  irregular  but  smoothly  undulating  surface, 
like  the  surface  of  a  relief  map. 

Rimose  :  abounding  in  chinks,  clefts,  or  cracks. 

Internal  structure  of  colony  (microscopic). 

Amorphous :  without  definite  structure  as  below  specified. 
Hyaline  :  clear  and  colorless. 

Homogeneous :  structure  uniform  throughout  all  parts  of  the 
colony. 


36 


LABORATORY  BACTERIOLOGY 


Homochromous :  color  uniform  throughout. 
Granulations  or  blotchings. 
Finely  granular. 
Coarsely  granular. 
Grumose :  coarser  than  the  preceding,  a  clotted  appearance, 

particles  in  clustered  grains. 
Moruloid:  having  the  character  of  a  morula  segmented,  by 
which  the  colony  is  divided  into   more   or  less  regular 
segments. 
Clouded :  having  a  pale  ground,  with  ill-defined  patches  of 
a  deeper  tint. 


4  5  6 

Fig.  4.    Types  of  colonies:    i,  cochleate ;  2,  amoeboid;  3,  rhizoid; 
4,  mycelioid  ;  5,  filamentous ;  6,  curled  structure. 

\,  Colony  marking  or  striping  (surface). 

Reticulate :  in  the  form  of  a  network  like  the  veins  of  a  leaf. 
Areolate :  divided  into  rather  irregular  or  angular  spaces 

by  more  or  less  definite  boundaries. 
Gyrose :  marked  by  wavy  lines  indefinitely  placed. 
Marmorated :  showing  faint,  irregular  stripes,  or  traversed 

by  veinlike  markings  as  in  marble. 
Rivulose :  marked  by  lines,  like  the  rivers  of  a  map. 
Riinose  :  showing  chinks,  cracks,  or  clefts. 
Filamentous  :  as  already  defined. 
Floccose :  composed  of  filaments  densely  placed. 
Curled:  filaments  in  parallel  strands,  like  locks  or  ringlets, 

as  in  agar  colonies  of  Bad.  anthracis. 


THE  EXA-MINATION  OF  PLATE  CULTURES 


37 


Edges  of  colonies. 

Entire  :  without  toothing  or  division. 

Undulate:  wavy. 

Repand :  like  the  border  of  an  open  umbrella. 

Erose :  as  if  gnawed,  irregularly  toothed. 

Lodate. 

Lobulate  :  minutely  lobate. 

Aurictdate :  with  earlike  lobes. 

Lacerate  :  irregularly  cleft,  as  if  torn. 

Ei?nbriate  :  fringed. 

Ciliate :  hairlike  extensions,  radiately  placed. 

Tufted. 

Filamentous  :  as  already  defined. 

Curled :  as  already  defined. 


Fig.  5.  Structure  of  colonies  :  i ,  conglomerate  colony  ;  2,  toruloid 
colony;  3,  alveolate  structure;  4,  grumose  in  center;  5,  moru- 
loid;  6,  clouded;  7,  reticulate ;  8,  marmorated;  9,  gyrose. 

6.  Optical  characters  (after  Shuttleworth). 

Transparent :  transmitting  light. 

Vitreous  :  transparent  and  colorless. 

Oleaginous :    transparent  and  yellow ;    olive   to  linseed  oil 

colored. 
Resinous :  transparent  and  brown,  varnish  or  resin  colored. 
Translucent :  faintly  transparent. 
Porcelaneous :  translucent  and  white. 
Opalescent :  translucent,   grayish  white   by  reflected  light, 

smoky  brown  by  transmitted  light. 


38 


LABORATORY  BACTERIOLOGY 


Nacreous :  translucent,  grayish  white,  with  pearly  luster. 

Sebaceous :  translucent,  yellowish  or  grayish  white. 

Butyrous :  translucent  and  yellow. 

Ceraceous :  translucent  and  wax  colored. 

Opaque. 

Cretaceous  :  opaque  and  white,  chalky ;  dull,  without  luster. 

Dull :  without  luster. 

Glistening  :  shining. 

Fluorescent. 

Iridescent, 


6  7  8  9 

Fig.  6.  Character  of  borders  of  colonies:  i,  entire;  2,  undulate; 
3,  repand ;  4,  lobate-lobulate  ;  5,  auriculate  ;  6,  lacerate ;  7,  fim- 
briate ;  8,  ciliate ;  9,  erose. 


THE  PREPARATION  OF  CERTAIN  SPECIAL  MEDIA     39 


EXERCISE  X 

THE  PREPARATION  OF  CERTAIN   DIFFERENTIAL  AND 
SPECIAL  MEDIA 

52.  In  studying  the  properties  of  bacteria  it  is  desirable  to 
cultivate  them  on  a  number  of  different  media.  Bouillon,  agar, 
and  gelatin  are  most  commonly  used,  but  others  are  neces- 
sary in  determining  the  cultural  peculiarities  and  important 
biochemic  properties  of  the  organism  in  question.  The  culti- 
vation of  bacteria  upon  these  media  may  be  regarded  in  the 
light  of  a  test  to  determine  the  presence  or  absence  of  certain 
properties  or  powers  possessed  by  the  bacterium  in  question  : 
thus,  for  example,  whether  the  species  in  hand  will  coagulate 
the  casein  in  milk,  produce  gas  in  media  containing  saccha- 
rose, grow  on  potato,  etc.  The  number  of  these  tests  which 
have  been  used  and  recognized  as  important  is  quite  large, 
but  in  a  short  course  only  those  possessed  of  special  differen- 
tial value  can  be  tried.  In  describing  a  new  species  or  iden- 
tifying any  of  the  carefully  described  ones,  it  is  important  to 
know  at  least  some  of  these  cultural  peculiarities  and  biochemic 
properties.  For  this  reason  it  is  necessary  to  learn  the  method 
of  preparation  and  the  use  of  certain  of  these  media.  The 
more  important  of  such  media  are  included  in  this  exercise. 

In  addition  to  the  above,  a  few  species  of  bacteria  require 
particular  kinds  of  media  for  their  diagnostic  or  most  differ- 
ential growth.  Among  these  are  the  specific  organisms  of 
glanders,  diphtheria,  and  tuberculosis.  The  preparation  of 
these  particular  media  will  be  considered  in  connection  with 
the  study  of  the  organisms  requiring  them. 

53.  Work  for  this  exercise.  Prepare  for  culture  media 
5  tubes  of  potato,  5  tubes  of  milk,  5  tubes  of  litmus  milk, 
5  tubes  of  glucose  agar,  5  tubes  of  glycerin  agar,  3  fermenta- 
tion and  5  small  test  tubes  of  bouillon  containing  glucose,  the 


40         LABORATORY  BACTERIOLOGY 

same  number  and  kinds  of  tubes  containing  lactose,  and  the 
same  containing  saccharose.  (The  agar  and  the  sugar-free 
bouillon  necessary  in  the  work  of  this  exercise  will  be  furnished 
by  the  instructor.) 

Read  carefully  the  paragraphs  in  the  text-books  on  the 
preparation  and  use  of  these  media. 

54.  Preparation  of  potato  for  a  culture  medium.  Select  3 
medium-sized  potatoes,  thoroughly  wash  and  rinse  in  boiled 
water,  and  cut  out,  with  a  cutter  made  for  this  purpose,  cylin- 
ders from  3  to  4  cm.  long  (oblong  rectangular  pieces  cut  with 
a  knife  will  do  quite  as  well).  Ordinarily  2  cylinders  can  be 
cut  from  each  potato.  These  can  be  cut  obliquely,  giving  2 
pieces  each.  All  of  the  skin  must  be  removed.  Wash  the 
potato  cylinders  in  cold  running  water  for  5  minutes  (a 
longer  time  is  preferable),  place  them  in  test  tubes  of  the 
proper  size  (large  or  small  according  to  size  of  cutter  used), 
and  add  about  i  cc.  of  water  to  each  tube.  Sterilize  them 
by  discontinuous  boiHng  or  steaming  for  20  minutes  each  day 
for  3  consecutive  days.     Wipe,  label,  and  store  in  locker. 

55.  Preparation  of  milk  for  a  culture  medium.  It  is  better 
that  the  cream  be  removed  from  the  milk  before  it  is  used. 
To  do  this  the  fresh  milk  (about  100  cc.)  is  placed"  in  a  beaker 
and  set  in  the  ice  box  for  from  10  to  15  hours.  Then  care- 
fully remove  the  cream.  It  is  well  to  filter  'the  milk  through 
a  thin  layer  of  absorbent  cotton  to  remove  any  masses  of 
cream  that  may  be  left.  The  reaction  should  be  tested,  and 
if  strongly  acid,  should  be  rejected  or  made  1.5%  acid  to 
phenol-phthalein  by  the  addition  of  n/io  sodium  hydrate. 
Distribute  the  skimmed  milk  in  small  test  tubes  (5  cc.  in  each) 
and  sterilize  by  discontinuous  steaming  in  the  same  manner  and 
for  the  same  length  of  time  as  the  potatoes.  Wipe,  label,  and 
store  the  tubes  in  locker. 

56.  Preparation  of  litmus  milk  for  a  culture  medium.  This 
is  prepared  the  same  as  the  milk  medium,  with  the  addition  of 
enough  of  an  aqueous  solution  of  Htmus  to  impart  a  decidedly 


THE  PREPARATION  OF  CERTAIN  SPECIAL  MEDIA     4I 

blue  color  to  the  milk.     Sterilize,  wipe,  label,  and  store  the 
same  as  the  milk.     The  litmus  solution  will  be  furnished. 

57.  Preparation  of  glucose  (grape  sugar)  agar.  Take  30  cc. 
of  agar  previously  prepared  (§  18),  liquefy,  and  add  1%  glu- 
cose while  hot.  After  thoroughly  mixing,  distribute  it  in  small 
sterile  test  tubes.  Sterilize,  wipe,  label,  and  store  the  same 
as  ordinary  agar. 

58.  Preparation  of  glycerin  agar.  Take  50  cc.  of  the  agar 
previously  prepared  and  add  5%  of  pure  (c.p.)  glycerin. 
Thoroughly  mix  it  with  the  agar,  after  which  distribute  it  in 
tubes.     Sterilize,  label,  and  store  as  ordinary  agar. 

59.  Preparation  of  glucose  bouillon.  This  is  used  in  the 
fermentation  tube.  Take  100  cc.  of  sugar-free  peptonized 
bouillon  (§  62)  and  add  i  gram  of  pure  grape  sugar  (glucose). 
After  it  is  dissolved  and  thoroughly  disseminated  through  the 
bouillon  by  stirring  or  pouring,  distribute  the  bouillon  in  3 
fermentation  tubes,  filling  completely  the  closed  branch  and 
the  open  bulb  about  half  full,  and  put  5  cc.  in  each  of  5  small 
sterile  test  tubes.  Sterilize  by  discontinuous  steaming  for  20 
minutes  each  day  for  3  consecutive  days.  The  tubes  should  be 
wiped,  labeled,  and  placed  in  the  locker  until  needed  for  use. 

60.  Preparation  of  lactose  bouillon.  This  is  prepared  by 
adding  i  %  of  pure  lactose  (milk  sugar)  to  the  peptonized 
sugar-free  bouillon.  It  is  necessary  that  the  bouillon  used 
does  not  contain  muscle  sugar.  After  adding  the  lactose, 
which  has  been  dissolved  and  thoroughly  mixed  in  a  few  cubic 
centimeters  of  the  bouillon,  distribute  in  fermentation  tubes 
and  small  test  tubes,  sterilize,  label,  and  store  the  same  as 
the  glucose  bouillon. 

61.  Saccharose  bouillon.  This  is  peptonized  sugar-free  bou- 
illon to  which  I  %  pure  saccharose  (cane  sugar)  has  been  added. 
It  is  prepared  from  bouillon  free  from  muscle  sugar  in  the 
same  manner  as  lactose  bouillon. 

62.  Preparation  of  sugar-free  bouillon.  Bouillon  prepared 
by  the  ordinary  method  usually  contains  small  quantities  of 


42  LABORATORY  BACTERIOLOGY 

muscle  sugar.  To  eliminate  this  the  following  method  has 
been  recommended.^  Beef  infusion  is  inoculated  in  the  even- 
ing with  a  rich  fluid  culture  of  some  acid-producing  organism 
{B,  coli  communis)  and  placed  in  the  incubator.  The  next 
morning  the  white  of  an  egg  is  added,  and  the. infusion  is 
boiled  and  filtered.  Peptone  and  salt  are  added  as  usual. 
It  is  boiled,  filtered  again,  distributed  in  tubes  or  flasks  as 
desired,  and  sterilized  the  same  as  bouillon  (§  12). 

63.  Preparation  of  acid  agar.  This  is  prepared  in  the  same 
manner  as  ordinary  agar  (§  18),  with  the  omission  of  the  sodium 
hydrate  in  the  bouillon  from  which  it  is  made. 

64.  Preparation  of  acid  glycerin  agar.  Add  5  %  glycerin  to 
acid  agar  before  sterilizing  it. 

65.  Preparation  of  acid  glycerin  bouillon.  This  is  prepared 
either  as  ordinary  bouillon  (§  12)  or  as  sugar-free  bouillon 
(§  62),  with  the  omission  of  the  alkali  and  the  addition  of 
5  %  c.p.  glycerin. 

66.  Preparation  of  blood  serum.  When  a  small  quantity  is 
sufficient  it  can  be  obtained  from  a  dog  aseptically.  The 
animal  is  properly  tied  on  the  operating  table,  etherized,  the 
skin  over  the  carotid  or  femoral  artery  is  thoroughly  dis- 
infected and  turned  back,  the  artery  exposed,  a  sterile  glass 
canula  inserted,  and  the  blood  collected  in  a  sterile  flask  by 
means  of  a  sterile  rubber  tube  attached  to  the  canula.  After 
the  serum  is  formed  it  can  be  drawn  off  with  a  sterile  pipette 
and  distributed  in  small  sterile  test  tubes  (5-7  cc.  in  each). 
It  is  well  to  set  the  liquid  serum  in  an  incubator  for  a  few 
days  to  test  its  sterility.  The  tubes  of  liquid  serum  are  inclined 
(the  same  as  agar)  and  placed  in  a  blood-serum  sterilizer,  or 
other  chamber,  in  which  the  temperature  can  be  raised  to  70° 
<^r  75°  C.  until  the  serum  has  set.     Store  in  a  cool  place. 

If  larger  quantities  of  the  blood  are  required,  it  is  more  con- 
venient to  collect  it  from  bleeding  animals  in  a  slaughterhouse. 

^  ^nx\\\v,  Jourjtal  of  Expe^'imenial  Medicine,  Vol.  II  (1897),  p.  543. 


THE  PREPARATION  OF  CERTAIN  SPECIAL  MEDIA     43 

In  this  case  it  is  often  necessary  to  sterilize  the  liquid  serum 
after  it  has  been  distributed  in  tubes.  This  can  be  done  in  a 
water  bath  at  62°  C.  for  2  hours  each  day  for  4  consecutive  days. 

67.  Preparation  of  Loefiler's  blood  serum.  This  consists  of 
I  part  neutral  bouillon  (prepared  from  meat)  containing  i  % 
grape  sugar  and  3  parts  liquid  blood  serum.  Mix  and  dis- 
tribute in  sterile  test  tubes,  incHne,  and  solidify  the  same  as 
blood  serum.  The  temperature  should  be  about  75°  C,  and 
the  exposure  will  be  necessarily  longer  than  for  the  pure  blood 
serum.  When  it  is  to  be  used  for  the  cultivation  of  diphtheria 
organisms  it  can  be  set  at  a  much  higher  temperature  (80°  to 
100°  C).     Label  and  store. 

68.  Preparation  of  egg  medium.  The  whole  egg  is  pref- 
erable. Carefully  break  the  shell  of  the  required  number 
(3,  6,  or  more)  of  fresh  eggs,  drop  the  entire  contents  into 
a  sterile  beaker,  and  carefully  stir  with  a  sterile  glass  rod,  care 
being  taken  to  avoid  air  bubbles.  After  the  egg  is  well  mixed 
it  is  poured  into  test  tubes  (6  to  10  cc.  in  each)  and  sterilized 
by  heating,  preferably  in  a  serum  water  bath  at  70°  C.  for 
from  4  to  5  hours  each  day  for  2  days.  After  steriUzation  the 
tubes,  if  test  tubes,  should  be  sealed.  Before  using,  add  a  few 
drops  of  sterile  water  or,  better  perhaps,  of  5  %  glycerin  or  of 
glucose  to  afford  sufficient  moisture.  This  is  most  used  at  the 
present  time  for  the  cultivation  of  tubercle  bacteria  (Dorset, 
Am,  Med.,  April  5,  1902). 

69.  Preparation  of  nitrate  bouillon.  Take  peptonized  bouillon 
200  cc.  Add  potassium  nitrate  (0.5%)  i  gram.  Dissolve  the 
nitrate  in  the  bouillon,  put  in  tubes,  and  sterilize  the  same  as 
bouillon. 

The  nitrate  of  sodium  or  ammonium  may  be  substituted  for 
that  of  potassium.  The  salt  may  be  added  in  the  proportion 
of  from  0.1  to  1%  to  meet  special  demands. 

For  other  methods  and  special  media,  see  text-books. 

70.  Grouping  of  culture  media.  For  convenience  in  reference 
and  assignment  of  media,  the  culture  media  most  commonly 


44  LABORATORY  BACTERIOLOGY 

employed  in  studying  and  differentiating  species  of  bacteria 
have  been  arranged  arbitrarily  in  groups.  Large  test  tubes 
containing  agar  and  gelatin  for  making  plate  cultures  are  not 
included  in  these  groups. 

Group  A,   Media  commonly  used. 

A  tube  of  bouillon. 

A  tube  of  sugar-free  bouillon. 

A  tube  of  agar. 

A  tube  of  gelatin. 

A  tube  of  milk. 

A  tube  of  litmus  milk. 

A  tube  of  potato. 

Group  B,  Media  favorable  to  determine  the  power  of  bac- 
teria to  ferment  the  sugars  with  the  formation  of  acids. 

A  tube  of  bouillon  containing  i  %  grape  sugar  (dextrose, 

glucose). 
A  tube  of  bouillon  containing  i  %  milk  sugar  (lactose). 
A  tube  of  bouillon  containing  i  %  cane  sugar  (saccharose). 

Group  C,  Media  favorable  for  determining  the  production 
of  gas. 

A  tube  of  agar  containing  i  %  grape  sugar. 
A  tube  of  agar  containing  i  %  milk  sugar. 
A  tube  of  agar  containing  i  %  cane  sugar. 

Group  D,  Media  and  tubes  favorable  for  approximate  gas 
analysis  and  the  determining  of  the  aerobic  or  anaerobic 
tendencies. 

Fermentation  tube  containing  i  %  grape-sugar  bouillon. 
Fermentation  tube  containing  i  %  milk-sugar  bouillon. 
Fermentation  tube  containing  i  %  cane-sugar  bouillon. 

The  fermentation  tubes  containing  bouillon  with  sugars  may  be 
substituted  for  the  media  containing  sugars  in  Groups  B  and  C  if 
desired. 


THE  PREPARATION  OF  CERTAIN  SPECIAL  MEDIA     45 

Group  E,  Media  either  necessary  for  or  especially  desir- 
able for  the  cultivation  or  differentiation  of  certain  pathogenic 
bacteria. 

Acid  agar  (in  small  tubes). 
Acid  glycerin  agar  (in  small  tubes). 
Acid  glycerin  bouillon  (in  small  tubes). 

Blood  serum  (dog)  solidified  at  70°  to  75°  C.  (ground-glass- 
capped  tubes). 
Loefifler's  blood  serum  (usually  small  tubes). 
Egg  medium  (Dorset). 


46  LABORATORY   BACTERIOLOGY 


EXERCISE  XI 

INOCULATING  SPECIAL  MEDIA  AND  EXAMINING 
CULTURES 

71.  Work  for  this  exercise.  Inoculate,  from  a  culture  fur- 
nished of  B.  proteus  vidgaris^  a  tube  of  potato,  one  of  milk, 
one  of  litmus  milk,  one  of  glucose  agar,  a  fermentation  and 
test  tube  of  glucose,  one  each  of  lactose  and  of  saccharose 
bouillon.     Label  each  and  place  in  the  incubator. 

Stain  a  preparation  with  alkaline  methylene  blue,  one  with 
carbol  fuchsin,  and  one  with  an  aqueous  solution  of  gentian 
violet  from  the  bouillon  and  agar  cultures  (§  47).  Make  a 
careful  comparison  of  the  preparations  and  note  any  difference 
in  the  appearance  of  the  bacteria  or  in  the  intensity  of  the 
stain.  Preserve  as  a  permanent  specimen,  to  accompany  the 
notes,  a  preparation  stained  with  each  of  the  dyes. 

Prepare  the  aqueous  solution  of  gentian  violet  (§  38). 

(P'or  methods  of  making  anaerobic  cultures,  see  Exercise 
XXXIX.) 

72.  The  inoculation  of  glucose  agar  to  determine  the  power 
of  the  organism  to  produce  gas.  Boil  the  tube  of  glucose  agar 
in  an  open  water  bath  until  it  is  liquefied,  then  cool  it  down  to  a 
temperature  of  40°  C.  and  inoculate  it  with  a  loopful  of  the  cul- 
ture, carefully  stir  the  agar  with  the  loop,  after  which  solidify 
it  as  quickly  as  possible.     Label  and  stand  in  the  incubator. 

73.  The  use  of  media  containing  the  sugars.  The  sugars  are 
employed  as  tests  to  determine  whether  or  not  the  bacteria  in 
question  will  ferment  them,  producing  acids.  Some  bacteria 
will  produce  gas  as  well  as  acids.  The  latter  is  determined  in 
the  sugar-agar  tubes. 

In  the  fermentation  tubes  we  can  determine  both  of  these 
properties  and  also  the  quantity  of  gas  set  free.     It  is  easier, 

1  Or  any  other  gas-producing  bacillus. 


INOCULATING   SPECIAL  MEDIA  47 

however,  to  determine  the  acid  and  gas  production  in  the  test 
tubes  than  to  use  the  fermentation  tubes,  and  it  is  cheaper. 
It  is  convenient,  therefore,  to  use  these  tubes  with  the  sugar 
media  as  follows  : 

1.  If  to  determine  the  power  of  the  organism  to  produce 
gas,  use  only  the  agar  tubes. 

2.  If  to  determine  the  power  of  the  organism  to  ferment 
sugars,  producing  acids,  use  only  test  tubes  of  bouillon. 

3.  If  to  determine  the  quantity  of  gas  produced  and  approxi- 
mately its  composition,  use  the  fermentation  tube.  In  this 
exercise  all  three  are  called  for. 


48  LABORATORY   BACTERIOLOGY 


EXERCISE   XII 

THE  EXAMINATION  OF  CULTURES  ON  SPECIAL  MEDIA, 
WITH  A  STUDY   OF  THE  GAS   PRODUCTION 

74.  As  certain  of  these  media  are  used  to  determine  the 
effect  of  the  bacteria  upon  them,  it  is  important  to  observe 
very  carefully  not  only  the  appearance  of  the  growth  of  the 
bacteria  but  also  their  effect,  if  any,  upon  the  medium  on 
or  in  which  they  are  growing.  This  is  especially  noticeable 
in  the  milk,  litmus  milk,  and  sugar  bouillon  cultures.  The 
changes  here  are  largely  due  to  the  action  of  the  bacteria  on 
the  sugars  or  their  power  to  produce  alkali. 

The  knowledge  of  the  powers  of  a  given  species  of  bacteria 
to  produce  gas  when  grown  in  a  medium  containing  sugar  is 
also  quite  important.  It  is  desirable  to  determine  both  the 
quantity  of  gas  and  its  relative  composition.  Chemical  analy- 
ses have  shown  that  in  all  cases  tested  the  gas  resulting 
from  the  fermentation  of  the  sugar  consists  of  a  mixture  of 
hydrogen  (H)  and  carbonic  acid  gas  (CO2),  with  mere  traces 
of  other  gases.  It  is  important  to  know  also  the  quantity  of 
gas  produced  with  the  various  sugars,  especially  glucose,  lac- 
tose, and  saccharose.  To  determine  simply  whether  an  organ- 
ism will  produce  gas,  it  is  only  necessary  to  inoculate  it  into 
tubes  of  liquid  agar  containing  the  various  sugars ;  but  if  the 
quantity  of  gas  is  to  be  determined,  the  fermentation  tube 
is  the  most  convenient  apparatus  to  use.  In  some  cases  the 
gas  formation  is  one  of  the  .most  striking  differential  proper- 
ties, as  will  be  seen  in  the  study  of  hog-cholera  and  typhoid 
bacilli. 

References.  For  a  discussion  of  the  gas  production  and  use 
of  the  fermentation  tube,  see  Smith,  Wilder  Quarter-Century  Book, 
1893^  P-  1^7  ;  for  the  chemical  formulae,  see  Novy,  Laboratory 
Work  in  Bacteriology,   1899. 


THE  EXAMINATION   OF  CULTURES  49 

75.  Work  for  this  exercise.  Examine  and  describe  the 
cultures  made  on  the  special  media  in  Exercise  XI. 

Examine  the  bacteria  on  the  potato  culture  microscopically 
(i)  in  the  fresh  condition  (hanging-drop  preparation)  and  (2) 
in  a  stained  (carbo-fuchsin)  cover-glass  preparation.  Describe 
the  appearance  of  the  bacteria  (§  35).  Examine  the  fermen- 
tation tubes  and  indicate  the  quantity  of  gas. 

76.  A  few  points  to  be  observed  in  studying  cultures  on 
special  media,  (a)  Potato.  Note  carefully  the  extent  and 
color  of  the  growth  and  its  consistence. 

(b^  Milk,  Note  whether  or  not  the  ge-neral  appearance  and 
odor  of  the  milk  have  been  changed,  and  observe  whether  the 
casein  has  been  coagulated,  giving  a  firm,  solid  coagulum,  or 
precipitated.  Is  the  coagulum  covered  with  a  liquid  (serum)? 
if  so,  is  it  clear  or  milky?  Is  there  any  appearance  suggestive 
of  saponification?  Determine  its  consistence,  chemical  reac- 
tion as  indicated  by  litmus  paper  (§  27),  and  give  as  descriptive 
a  name  as  possible  to  its  odor. 

{c)  Litmus  milk.  Note  especially  whether  there  has  been 
any  change  in  color  since  inoculation.  Observations  similar 
to  those  on  the  plain  milk  should  also  be  made. 

(^/)  Glucose  agar.  Note  the  character  and  number  of  col- 
onies within  the  agar,  and  the  presence,  if  any,  of  gas  bubbles. 
Are  there  few  or  many  of  them? 

{e)  Bouillon  containifig  sugars  in  test  tubes.  Note  carefully 
the  appearance  of  the  bouillon,  but  especially  its  chemical 
reactions  as  indicated  by  the  litmus  paper  (§27). 

(/)  Bouillon  containing  sugars  in  fermentation  tubes.  Ob- 
serve the  character  of  the  growth  in  each  tube  (whether  the 
liquid  is  faintly  or  heavily  clouded,  turbid,  contains  flakes,  etc.), 
—  in  (i)  the  open  bulb  and  in  (2)  the  closed  branch  of  the 
fermentation  tube.  Note  the  presence  or  absence  of  a  mem- 
brane on  the  surface  of  the  liquid  in  the  open  bulb.  Is  there 
a  sediment  in  the  bottom  of  the  tube?  If  so,  describe  its 
general  appearance  and  consistence.     Note  the  presence  or 


50  LABORATORY  BACTERIOLOGY 

absence  of  gas  in  the  closed  branch.  Indicate  the  quantity 
and  note  its  rate  of  formation  from  time  to  time.  Test  the 
reaction  of  the  liquid  with  litmus  paper. 

The  fermentation  tubes  are  also  used  to  enable  one  to 
determine  the  quantity  and  kinds  of  gases  produced  and  the 
aerobic  or  anaerobic  tendencies  of  the  organism. 

In  studying  the  cultures  in  the  fermentation  tubes  they 
should  be  examined  each  day  and  the  quantity  of  gas  indi- 
cated. Note  the  bubbles  of  gas  rising  through  the  Hquid  to 
the  top.  When  the  gas  production  has  ceased,  the  Hquid 
begins  to  clear  near-  the  surface  in  the  closed  branch.  The 
final  record  should  not  be  made  until  this  occurs.  The  reac- 
tion of  the  culture  should  be  determined  and  noted  at  this 
and  the  next  exercise.  Explain  the  chemical  formulae  for  the 
production  of  the  gas,  and  if  the  reaction  changes,  give  the 
explanation. 

77.  Determination  of  the  quantity  of  gas.  It  is  desirable  to 
determine  the  quantity  of  gas  collected  in  the  closed  branch 
in  terms  of  the  capacity  of  the  tube.  To  do  this,  measure 
the  length  of  the  closed  branch  and  the  length  of  that  portion 
of  the  tube  filled  with  gas.  Thus,  if  the  length  of  the  tube  is 
lo  cm.  and  the  length  of  the  portion  filled  with  gas  is  3  cm., 
the  gas  fills  three  tenths  of  the  branch.  This  cannot  be  deter- 
mined until  the  gas  formation  has  ceased,  which  sometimes 
requires  several  (4  to  6)  days.  The  closed  branch  of  the  fer- 
mentation tube  should  be  straight  and  the  connecting  part  of 
the  tube  should  be  narrow."  If  the  tube  stands  too  long  before 
the  quantity  of  gas  is  determined,  some  of  it  is  liable  to  be 
absorbed. 

78.  To  determine  the  ratio  of  CO2  to  H  in  the  gas  produced. 
This  can  be  approximately  determined  by  the  use  of  caustic 
soda.  Remove  the  plug  from  the  fermentation  tube  and  fill 
the  open  bulb  with  a  2  %  solution  of  caustic  soda.  Place  the 
thumb  tightly  over  the  open  end  of  the  tube  and  tip  it  up  so 
that  the  gas  will  pass  through  the  liquid  and  come  into  the 


THE  EXAMINATION   OF  CULTURES  5  I 

open  bulb.  It  is  then  returned.  This  should  be  repeated 
several  times.  Remove  the  thumb  when  the  open  bulb  is 
full,  and  the  liquid  will  rush  up  into  the  closed  branch  to  fill 
the  space  occupied  by  the  CO2  which  has  been  absorbed  by 
the  caustic  soda.  Measure  the  portion  of  the  tube  first  occu- 
pied with  gas  and  now  filled  with  the  liquid.  This  wall  indi- 
cate the  quantity  of  CO2.  The  remainder  of  the  gas  is  H. 
(There  are  also  traces  of  other  gases.)  Its  explosive  property 
can  be  tested  by  filHng  the  open  bulb  with  water,  covering  it 
with  the  thumb  and  again  bringing  the  gas  to  the  open  bulb, 
holding  it  close  to  a  flame,  and  removing  the  thumb.  A  dis- 
tinct explosion  will  be  heard. 

The  ratio, of  CO2  to  H  can  be  determined  from  the  meas- 
urements. Thus  the  total  amount  of  gas  in  the  closed  branch 
=  5  cm.  The  amount  absorbed  (CO2)  =  2  cm.  The  remain- 
ing gas,  or  3  cm.,  =  H.  The  ratio  of  CO2  to  H  is,  therefore, 
as  2  :  3,  or  CO2  :  H  :  :  2  :  3. 


52  LABORATORY  BACTERIOLOGY 

EXERCISE  XIII 

THE   EXAMINATION   OF  CULTURES   {continued) 

79.  Work  for  this  exercise.  Reexamine  the  cultures  made 
on  special  media  and  make  notes  on  all  changes  which  have 
occurred  in  their  appearance. 

Examine  microscopically  in  hanging-drop  preparations  the 
bacteria  from  the  glucose  bouillon  culture. 

Make  a  stained  cover-glass  preparation  from  the  milk 
culture.     Stain  with  carbol  fuchsin. 

Reexamine  all  of  the  cultures  previously  made  and  make 
careful  notes  of  any  changes  that  have  occurred  in  their 
appearance. 

Measure  the  gas  in  the  fermentation  tubes  and  determine 
the  ratio  of  CO2  to  H. 

80.  Making  cover-glass  preparations  from  milk  cultures. 
Spread  as  thin  a  film  of  the  milk  culture  as  possible  on  the 
cover  glass  and  allow  it  to  dry  in  the  air.  Immerse  the  prep- 
aration in  a  watch  glass  or  other  receptacle  containing  a  few 
cubic  centimeters  of  ether  and  absolute  alcohol  in  equal  parts, 
which  dissolves  out  the  fat  and  fixes  the  film  to  the  cover  glass 
at  the  same  time.  Then  remove  and,  after  the  ether  and  alco- 
hol have  evaporated,  stain  as  usual.  The  amount  of  albumen 
in  the  milk  will  usually  cause  a  heavy  background,  which  will 
require  decolorizing  with  alcohol  or  weak  acetic  acid. 


THE  CLASSIFICATION  OF  BACTERIA  53 

EXERCISE    XIV 

THE  CLASSIFICATION  OF  BACTERIA 

81.  The  term  "  bacteria  "  is  a  general  and  popular  one  used 
to  designate  a  large  group  of  microscopic  plants,  the  Schizo- 
mycetes.  These  organisms,  which  are  widely  distributed  in 
nature,  have  been  classified  into  a  certain  few  families  and 
genera  most  of  which  have  a  large  number  of  species.  Many 
of  these  species  have  been  described,  but  there  are  many  which 
have  not.  In  classifying  the  bacteria,  the  genera  are  based  on 
morphologic  characters  ;  while,  as  a  rule,  the  species  are  deter- 
mined by  means  of  their  biochemic,  physiologic,  or  pathogenic 
properties.  Several  systems  of  classification  have  been  pro- 
posed, but  the  one  which  seems  to  be  the  most  satisfactory  is 
by  Migula.  This  classification  utilizes  the  morphology  to  such 
good  advantage  that  its  adoption  seems  desirable.  It  requires, 
however,  some  serious  changes  in  the  accustomed  nomen- 
clature ;  but  this  is  true  of  any  logical  system.  The  restoration 
of  the  genus  Bactefiiim  and  the  assigning  to  it  of  all  non- 
motile,  rod-shaped  organisms  change  the  genus  of  some  of 
our  most  common  pathogenic  bacteria  from  Bacillus  to  Bac- 
terium. The  most  conspicuous  of  these  are  the  bacilli  of 
tuberculosis,  glanders,  and  diphtheria,  all  of  which  are  placed 
in  Migula's  classification  in  the  genus  Bacterium,  The  famiUes 
and  genera  recognized  by  him  are  appended.  (See  lecture  notes 
and  text-books  on  the  classification  and  morphology  of  bacteria.) 

82.  Work  for  this  exercise.  Read  the  references  on  the 
morphology  and  classification  of  bacteria. 

Reject  all  the  cultures  made  and  clean  the  tubes  and  Petri 
dishes  [§  3,  (/)]. 

Inoculate  a  tube  of  bouillon  (from  cultures  which  will  be 
furnished)  with  each  of  the  following  genera  of  bacteria  :  (i)  a 
streptococcus,  (2)  a  micrococcus,  (3)  a  sarcina. 


54  LABORATORY    BACTERIOLOGY 

83.  Migula's  classification  of  bacteria. 

FAMILIES 

L  Cells  globose  in  a  free  state,  not  elon- 
gating in  any  direction  before  divi- 
sion into  I,  2,  or  3  planes  .  .  .  .  i.  CoccACEiE 
II.  Cells  cylindrical,  longer  or  shorter,  and 
only  dividing  in  i  plane,  and  elon- 
gating to  twice  the  normal  length 
before  the  division. 

1.  Cells  straight,  rod-shaped,  without 

sheath',  nonmotile,  or  motile   by 

means  of  flagella 2.  BACTERiACEiE 

2.  Cells  crooked,  without  sheath     .     .3.  SpiRiLLACEiE 

^  ,,    .     1      J  .         ,      ^,  (4.  Chlamydobacte- 

3.  Cells  mclosed  m  a  sheath      .     .    4    ^        T3TAr>T:.:r^ 

i  RIACE^ 

4.  Cells  destitute  of  a  sheath,  united 

into  threads,  motile  by  means  of 

an  undulating  membrane    .     .     .5.   Beggiatoace^ 

1.  GENERA   BELONGING    TO    THE   FAMILY   COCCACE^ 

Cells  without  organs  of  motion. 

a.  Division  in  i  plane i.  Streptococcus 

b.  Division  in  2  planes 2.  Micrococcus 

c.  Division  in  3  planes 3.  Sarcina 

Cells  with  organs  of  motion. 

a.  Division  in  2  planes    .     .  -  .     .     .     .4.  Planococcus 

b.  Division  in  3  planes 5.  Piano  sarcina 

2.  GENERA  BELONGING  TO  THE  FAMILY  BACTERIACE^E 

Cells  without  organs  of  motion     .     .     .     .   i .  Bacterium 
Cells  with  organs  of  motion  (flagella). 

a.  Flagella  distributed  over  the  whole 

body 2.  Bacillus 

b.  Flagella  polar 3.  Psetidomonas 

3.  GENERA   BELONGING   TO   THE   FAMILY   SPIRILLACEiE 

Cells  rigid,  not  snakelike  or  flexuous. 

a.  Cells  without  organs  of  motion      .     .1.  Spirosoma 


THE   CLASSIFICATION   OF  BACTERIA  55 

b.  Cells  with  organs  of  motion  (flagella). 
(i)  Cells  with  I,  very  rarely  2-3,  polar 

flagella 2.  Microspira 

(2)   Cells  with  polar  flagella,  in  tufts 

of  from  5  to  20 3.  Spirillum 

Cells  flexuous 4-  SpirochcBta 

4.   GENERA    BELONGING    TO    THE   FAMILY    CHLAMYDO- 
BACTERIACEiE 

I.  Cell  contents  without  granules  of  sulphur. 

a.  Cell  threads  unbranched. 

(i)  Cell  division  always  only  in  i  plane  i .  Streptothrix 
(2)  Cell  division  in  3  planes  previous 
to  the  formation  of  conidia. 
(a)  Cells  surrounded  by  a  very 
delicate,    scarcely     visible 

sheath  (marine)  .     .     .     .2.  Phragmidiothrix 
(J?)  Sheath    clearly     visible    (in 

fresh  water) 3.  Crenothrix 

b.  Cell  threads  branched  (pseudo- 

branches)      4-  Cladothrix 

II.  Cell  contents  containing  sulphur  gran- 

.  ules 5.  Thiothrix 

5.  GENERA  BELONGING  TO  THE  FAMILY  BEGGIATOACEiE 

Only  one  genus  known  {Beggiatoa  Trev.),  which  is  scarcely 
separable  from  Oscillaria.     Character  as  given  under  the  family. 

Of  these  genera  Streptococcus,  Micrococcus,  Bacterium, 
Bacillus,  Microspira,  and  Spirillum  contain  the  most  important 
of  the  pathogenic  bacteria.  The  familiar  genus  Staphylococcus 
of  older  classifications  is  included  in  the  genus  Micrococcus  by 
Migula.  It  is  important  that  the  distinguishing  characters  of 
these  genera  be  thoroughly  learned. 

References.  Migula,  Die  natiirlichen  Pflanzenfamilien,  Liefe- 
rung  129,  Leipsic,  1896.  Migula,  System  der  Bakterien,  1897. 
Fischer,  Jahrbiicher  fiir  wissenschaftliche  Botanik,  Band  XXVII, 
Erstes  Heft. 


S6  LABORATORY  BACTERIOLOGY 


EXERCISE   XV 

THE  MORPHOLOGY  OF  STREPTOCOCCUS,  MICROCOCCUS, 
AND    SARCINA 

84.  Genera  among  bacteria  are  based  on  the  gross  mor- 
phology of  the  organisms.  This  is  very  largely  true  of  all  clas- 
sifications. It  is  highly  important,  therefore,  that  the  generic 
characters  should  be  thoroughly  learned.  While  the  descrip- 
tive differences  between  a  micrococcus  and  a  bacterium  seem 
to  be  clear,  there  are  many  organisms  where  it  is  not  so  easy 
to  decide  in  which  genus  to  place  them.  The  almost  constant 
appearance  of  unexpected  bacteria  in  septic  infections  and  in 
diseased  organs  renders  it  exceedingly  desirable  that  one 
should  understand  the  fundamental  elements  of  classification. 
We  must  remember  that  the  problems  of  the  practitioner  are 
not  all  centered  about  known  pathogenic  forms  like  the  organ- 
isms of  tuberculosis  and  diphtheria ;  but  they  have  to  do  with 
a  great  host  of  infecting  bacteria,  of  which  we  know  as  yet  but 
very  little. 

85.  Work  for  this  exercise.  Carefully  describe  each  of 
the  bouillon  cultures  made  in  Exercise  XIV. 

Prepare  and  examine  a  hanging-drop  preparation  from  each 
of  the  cultures,  and  describe  the  appearance  (form)  of  the 
organisms  in  each.  Indicate  the  morphologic  characters  by 
which  each  genus  can  be  differentiated  from  the  others. 

Make  a  cover-glass  preparation  from  each  culture  and  stain 
with  an  aqueous  solution  of  methyl  violet  (§  38).  Make 
a  careful  microscopic  examination  of  each  preparation  and 
describe  the  bacteria  in  each. 

Make  careful  notes  on  the  appearance  of  the  bacteria  in  each 
preparation  and  preserve  a  specimen  of  each  to  accompany 
the  notes. 


MORPHOLOGY  57 

Inoculate  a  tube  of  bouillon  from  cultures  (furnished)  of 
each  of  the  following  genera  of  bacteria  :  (i)  a  bacterium,  (2)  a 
bacillus  (3  cults),  (3)  a  spirillum. 

(Select  species  of  bacilli  that  illustrate  slow  and  rapid 
motility  and  spores.) 


S8  LABORATORY  BACTERIOLOGY 

EXERCISE   XVI 

THE  MORPHOLOGY  OF  BACILLUS 

86.  The  bacilli  which  are  to  be  studied  in  this  exercise 
exhibit  in  addition  to  the  rod-shaped  bodies  the  essential  mor- 
phological variations  of  this  genus,  viz.  number  of  flagella 
and  spores.  The  two  latter  are  held  in  common  with  the 
bacterium.  The  staining  of  the  organs  of  locomotion  (flagella) 
and  the  spores  will  be  taken  up  in  separate  exercises. 

87.  Work  for  this  exercise.  Carefully  examine  and  study 
the  cultures  made  in  Exercise  XV,  following  the  directions 
given  for  the  examination  of  cultures  and  preparations  in  that 
exercise. 

Measure  carefully  with  the  filar  micrometer  the  length  and 
thickness  of  8  individual  bacteria  in  one  of  the  stained  prepa- 
rations. Record  the  measurements  in  microns  (written  /a). 
(For  the  use  of  the  micrometer,  see  Appendix,  also  chapter 
on  magnification  and  micrometry  in  The  Microscope,  by  Pro- 
fessor S.  H.  Gage.) 

State  fully  in  the  notes  the  generic  characters  of  the  genus 
Bacillus, 

Inoculate  a  tube  of  bouillon  and  one  of  agar  from  each  of 
the  cultures  (which  will  be  furnished)  of  a  Bacterium  and  of 
a  Spirillum, 

Inoculate  a  tube  of  agar  with  B,  subtilis  for  Exercise  XVIII. 


MORPHOLOGY  59 

EXERCISE   XVII 

THE  MORPHOLOGY  OF  BACTERIUM  AND  SPIRILLUM 

88.  Work  for  this  exercise.  Examine  very  carefully  and 
describe  fully  the  cultures  of  the  Bacterium  and  of  the  Spiril- 
lum made  in  the  last  exercise. 

Make  and  examine,  microscopically,  hanging-drop  and 
stained  cover-glass  preparations  from  each  of  the  cultures. 
Describe  the  appearance  of  the  individual  bacteria  in  each. 

Make  a  drawing  magnified  looo  diameters  of  a  few  indi- 
viduals from  each  of  the  stained  preparations  from  the  agar 
cultures. 

State  fully  the  morphology  of  these  two  genera  and  mention 
the  differential  characters  between  the  genera  Bacillus  and 
Bacterimn, 

Inoculate  a  tube  of  agar  with  B,  cholerce  suis  or  B,  typhosus 
for  flagella  staining  in  Exercise  XIX. 

89.  Making  drawings  of  bacteria  with  a  definite  magnifica- 
tion. In  measuring  the  bacteria  we  obtain  the  dimensions  in 
microns  or  in  units  of  i/iooo  of  a  millimeter.  In  making  a 
drawing,  therefore,  showing  them  magnified  looo  diameters, 
it  is  simply  necessary  to  represent  each  micron  by  i  millimeter. 
Thus,  if  the  organism  is  2.5  /i  in  length  and  i  fx  broad,  the 
drawing  should  be  2.5  mm.  long  and  i  mm.  broad.  If  the 
drawing  is  to  represent  the  organism  magnified  500  diameters, 
then  each  micron  should  be  represented  by  0.5  mm.  For  this 
purpose  a  metric  rule  and  a  pair  of  dividers  are  necessary. 


60         LABORATORY  BACTERIOLOGY 

EXERCISE   XVIII 

STAINING   SPORES 

90.  In  certain  species  of  bacteria  and  under  suitable  condi- 
tions there  appear  within  the  bacteria  highly  refractive  bodies 
known  as  spores.  The  formation  of  spores  is  restricted  to  cer- 
tain species.  The  spores  are  oval  in  form,  and  in  old  cultures 
they  may  often  be  found  outside  of  the  bodies  of  the  organisms 
which  produce  them.  They  possess  the  power  of  resisting  dry- 
ing, heat,  and  unfavorable  environment  much  longer  than  the 
bacilli  themselves.  They  do  not  stain  by  the  usual  methods 
employed  in  staining  bacteria,  so  special  methods  are  required. 
Several  processes  have  been  proposed,  but  the  one  here  given 
seems  to  be  quite  as  efificient  as  any  of  the  others. 

Bacillus  subtilis,  or  the  hay  bacillus,  is  one  of  the  most 
widely  distributed  species  of  bacteria.  It  develops  spores 
which  can  be  readily  detected  either  in  fresh  or  stained  prepa- 
rations from  cultures. 

References.     Methods  for  staining  spores  in  text-books. 

91.  Work  for  this  exercise.  Examine  and  carefully  describe 
the  culture  of  Bacillus  subtilis  made  in  Exercise  XVI. 

Make  a  hanging-drop  preparation  from  the  bouillon  and  one 
from  the  agar  culture  and  examine  them  microscopically. 
Describe  the  bacilli  and  observe  carefully  the  appearance  of 
the  spores  both  within  and  without  the  organisms. 

Make  a  cover-glass  preparation  from  each  culture  and  stain 
with  alkaline  methylene  blue.  Examine  carefully  and  note  the 
appearance  of  spores  which  remain  unstained.  Make  a  draw- 
ing of  a  few  of  the  bacteria  containing  spores. 

Make  a  few  (about  3)  cover-glass  preparations  and  stain 
them  for  spores. 


STAINING   SPORES  6l 

Prepare  water  suspensions  for  staining  flagella  as  described 
in  (§  95). 

92.  A  method  for  staining  spores.  Make  a  cover-glass 
preparation,  dry,  and  flame  as  already  described.  Take  the 
preparation  by  the  edge  with  the  fine  forceps,  cover  the  film 
surface  with  carbol  fuchsin,  and  hold  the  preparation  over  the 
gas  flame  until  steam  is  given  off;  then  remove  it  for  a  few 
seconds  and  heat  again.  Repeat  the  heating  three  or  four 
times.  After  the  stain  has  acted  for  from  3  to  5  minutes,  rinse 
the  preparation  in  water  and  decolorize  it  by  immersing  it  in 
a  watch  glass  containing  about  3  cc.  of  1%  solution  of  sul- 
phuric acid  or  95%  alcohol.  After  about  one  half  minute 
remove  the  preparation  and  rinse  it  thoroughly  in  water.  If  it 
is  not  decolorized,  repeat  the  bleaching  process.  This  removes 
the  coloring  matter  from  the  bodies  of  the  bacteria,  but  leaves 
it  in  the  spores.  After  thoroughly  washing  the  preparation, 
counterstain  it  with  a  saturated  aqueous  solution  of  methylene 
blue  for  about  30  seconds,  rinse  in  water,  and  examine.  The 
spores  should  be  stained  red  (with  the  fuchsin)  and  the  rest 
of  the  organism  should  be  colored  blue. 

There  is  a  very  satisfactory  method  recommended  by  MoUer. 
For  this  and  other  methods  for  staining  spores,  see  text-books 
on  bacteriology. 


62  LABORATORY  BACTERIOLOGY 

EXERCISE   XIX 

STAINING  THE  FLAGELLA  ON  MOTILE  BACTERIA 

93.  The  motile  bacteria  are  provided  with  a  variable  num- 
ber of  long,  hairlike  appendages  or  flagella.  These  are  invisi- 
ble in  the  fresh  preparation,  and  they  do  not  stain  by  the 
ordinary  methods.  By  special  staining  processes,  however,  their 
presence  can  be  detected.  Several  methods  have  been  pro- 
posed for  staining  these  filaments,  but  nearly  all  of  them  are 
based  on  the  use  of  a  mordant.  Curiously  enough  the  value 
of  each  of  these  methods  seems  to  rest  largely  on  the  skill  of 
the  individual  using  them,  as  some  workers  succeed  with  one 
method  while  others  fail  with  it  but  obtain  excellent  results 
with  one  of  the  other  processes.  Although  the  flagella  are 
known  to  be  the  organs  of  locomotion,  they  do  not  seem  to  be 
of  any  special  morphological  value  in  differentiating  closely 
related  species.  They  are,  however,  elements  in  the  structure  of 
motile  bacteria,  and  their  demonstration  is  much  to  be  desired. 

References.  Chapters  on  staining  flagella  in  standard  text- 
books. Moore,  A  Review  of  the  Methods  for  Staining  Flagella 
on  Motile  Bacteria,  Am.  Monthly  Mic.  Journal,  Vol.  XII  (1891), 
p.  15.  Moore,  The  Character  of  Flagella,  etc..  Wilder  Quarter- 
Century  Book,  p.  339.  Loeffier,  Centralblatt  fiir  Bakteriologie, 
etc.,  Bd.  VI  (1889),  S.  209.  Ferrier,  Achives  de  M^d.  Exp.  et 
d'Anat.  pathologique,  T.  VII  (1895),  p.  58.  Van  Ermengem, 
reviewed  in  Centralblatt  fiir  Bakteriologie,  etc.,  Bd.  XV  (1894), 
No.  24.     Johnston  and  Mack,  Am.  Med.,  Vol.  VII,  p.  754. 

94.  Work  for  this  exercise.  Make  a  cover-glass  preparation 
from  the  growth  on  the  agar  culture  of  Bacillus  cholercB  suis 
made  in  Exercise  XVII  and  stain  it  with  carbol  fuchsin. 
Preserve  this  to  compare  with  preparations  stained  for  the 
purpose  of  demonstrating  the  flagella. 

Clean  about  10  cover  glasses  after  the  special  method  for 
flagella  staining  (§  4).     Make  about  5  films  on  these  from  the 


STAINING  THE   FLAGELLA  63 

water  suspension  prepared  at  the  last  exercise  and  stain  for 
flagella.  Use  Johnston's  and  Mack's  modified  method,  with 
Loeffler's  mordant  and  stain.  The  films  should  not  be  heated 
either  before  or  during  the  staining  process,  but  if  it  does  not 
succeed,  Loeffler's  process  may  be  tried. 

95.  Johnston's  and  Mack's  modified  method,  (a)  Preparation 
of  films.  Make  a  culture  of  the  organism  to  be  stained  on  slant 
agar  and  incubate  for  from  18  to  24  hours.  Prepare  a  tube  with 
6  to  8  cc.  of  sterile  water,  and  keep  it  in  the  incubator  until 
it  is  of  the  same  temperature.  With  a  sterile  platinum  loop 
scrape  away  some  of  the  growth  from  the  agar  surface,  using 
care  not  to  remove  any  of  the  agar,  and  rinse  it  off  carefully 
in  the  tube  of  water  previously  prepared  for  this  purpose. 
There  should  be  enough  to  impart  to  the  water  a  faint  cloudi- 
ness. This  should  be  done  in  a  warm  room  and  with  con- 
siderable care.  Replace  this  tube  in  the  incubator  and  the 
bacilli  will  distribute  themselves  quite  evenly  through  the  water, 
but  any  clumps  or  masses  settle  to  the  bottom.  The  tube  is 
left  in  the  incubator  for  from  2  to  3  days  before  preparing 
the  films.  Place  a  tray  of  properly  prepared  cover  glasses  in 
the  incubator  to  warm ;  then,  still  in  the  warm  room,  with  a 
platinum  loop  put  a  drop  or  two  from  the  tube  on  each  cover 
(§  4).  Replace  the  tray  in  the  incubator  until  the  water  has 
evaporated,  when  the  films  are  ready  to  stain.  The  films 
require  no  fixing  other  than  by  the  mordant.  The  small 
amount  of  organic  matter  in  films  prepared  in  this  way  gives 
but  Httle  background  when  stained. 

(b)  Mordants  and  staifis.  Different  mordants  and  staining 
solutions  may  be  used.  Those  of  Loeffler  or  Pitfield  give  the 
most  uniform  and  satisfactory  results. 

loeffler's  mordant 

Twenty  per  cent  aqueous  solution  tannic  acid  .  .  10  cc. 
Saturated  aqueous  solution  iron  sulphate  .  .  .  5  cc. 
Saturated  alcoholic  solution  basic  fuchsin    .     .     .     i  cc. 

Mix,  let  stand  two  or  three  hours,  and  filter. 


64         LABORATORY  BACTERIOLOGY 

Tannic  acid  solution  should  be  freshly  prepared,  but  the 
iron  sulphate  solution  is  better  if  it  stands  until  it  begins  to  turn 
brownish  by  oxidation,  but  it  should  not  be  too  old.  If  when 
this  mordant  is  used  it  gives  a  precipitate,  filter  again.  When 
properly  prepared  it  should  have  much  the  same  color  as  a 
solution  of  hematoxylin. 

loeffler's  stain  (ziehl's  carbol  fuchsin) 

Saturated  alcoholic  solution  basic  fuchsin   ...     2.5  cc. 
Five  per  cent  carbolic  acid 20     cc. 

If  not  clear,  add  fuchsin  solution  drop  by  drop  until  it  clears, 
then  filter. 

pitfield's  mordant 

Ten  per  cent  aqueous  solution  tannic  acid  ...  10  cc. 

Saturated  aqueous  solution  mercuric  chloride  .     .  5  cc. 

Saturated  aqueous  solution  potassium  alum      .     .  5  cc. 

Ziehl's  carbol  fuchsin 5  cc. 

Mix,  let  stand  two  or  more  hours,  and  decant  clear  fluid  or 
filter. 

pitfield's  stain 

Saturated  aqueous  solution  potassium  alum     .     .   10  cc. 
Saturated  alcoholic  solution  gentian  violet  .     .     .     2  cc. 

Mix,  let  stand  two  or  more  hours,  and  filter. 

Saturated  alcoholic  solution  of  methyl  violet,  basic  fuchsin, 
or  methylene  blue  can  be  substituted  for  gentian  violet  in  the 
above  formula  with  equally  good  results. 

With  a  pipette  apply  to  a  film  all  the  mordant  the  cover  glass 
will  hold,  allow  it  to  act  two  or  three  minutes  without  heating, 
and  rinse  thoroughly  in  clean  water  ;  apply  the  stain  in  the  same 
manner,  allow  it  to  act  two  or  three  minutes  without  heating, 
rinse  well,  and  examine  in  water,  or  dry  and  mount  in  balsam. 

All  these  solutions  should  be  freshly  prepared.  Consider- 
able care  in  washing  after  the  mordant  will  be  well  repaid  by 
insuring  a  cleaner  background. 


STAINING  THE   FLAGELLA      '  6$ 

Around  the  edge  where  the  drop  was  apphed  to  the  cover 
glass  a  heavy  hne  of  bacteria  will  be  found,  and  if  the  right 
amount  of  culture  was  added  to  the  water,  many  will  be  found 
scattered  within  the  ring,  some  of  them  isolated  so  they  can 
be  easily  studied.  A  little  practice  with  this  will  enable  one 
to  make  good  preparations  altogether  free  from  background. 

96.  Staining  the  flagella  by  Loefiler*s  method.  Place  2  loop- 
fuls  of  sterilized  distilled  water  or  normal  salt  solution  on  the 
center  of  the  cover  glass.  Gently  touch  the  surface  growth 
on  the  agar  culture  with  the  end  of  the  platinum  needle  and 
immerse  it  in  the  water  on  the  cover  glass  without  spreading 
the  drop.  The  impregnated  needle  should  carry  bacteria 
enough  for  3  or  4  preparations.  Then  place  the  tray  of  cover 
glasses  in  the  incubator  to  dry.  The  bacteria  become  dissemi- 
nated throughout  the  water  by  means  of  their  power  of  loco- 
motion.    When  dry  they  are  ready  for  the  staining  treatment. 

The  bacteria  are  fixed  to  the  cover  glass  by  holding  them, 
film  upward,  between  the  thumb  and  forefinger,  over  a  gas 
flame  for  about  a  minute.  They  are  then  treated  with  the 
following  mordant. 

Tannic  acid,  20  %  solution 10  cc. 

Sulphate  of  iron,  saturated  solution 5  cc. 

Fuchsin,  saturated  alcohoUc  solution      .     .     .     .     i  cc. 

This  should  be  filtered  before  using. 

Place  the  fixed  cover-glass  preparation  in  a  large  test  tube, 
cover  it  with  the  mordant,  and  carefully  heat  over  a  gas  flame 
or  in  a  water  bath  until  steam  is  given  off.  Allow  the  mordant 
to  act  for  from  3  to  5  minutes.  Then  remove  the  cover  glass 
with  a  bent  wire  loop  and  fine  forceps  and  thoroughly  rinse  it 
in  water.  Then  place  it  in  a  similar  tube  and  cover  with 
carbol  fuchsin  for  staining.  Heat  this  as  the  mordant  was 
heated  and  allow  the  stain  to  act  for  from  5  to  10  minutes. 
Remove  the  cover  glass  as  before  and  thoroughly  rinse  in  water. 
If  the  stain  is  too  deep,  decolorize  by  rinsing  the  preparation 


66  LABORATORY  BACTERIOLOGY 

for  a  few  seconds  in  alcohol  and  again  in  water.  It  is  then 
ready  for  the  microscopic  examination  in  water,  or  it  may  be 
allowed  to  dry  and  then  be  mounted  in  balsam.  If  the  first 
preparation  fails,  add  2  drops  of  a  10%  solution  of  sulphuric 
acid  to  the  mordant. 

The  flagella  should  appear  as  fine,  hairlike  appendages  radi- 
ating from  the  bacteria. 

97.  Staining  the  flagella  by  Van  Ermengem*s  method.  The 
films  are  prepared  as  described  above.  Three  solutions  are 
necessary. 

SOLUTION   A    (fixing   BATH) 

Osmic  acid,  2  %  solution i  part 

Tannin,  10-25  %  solution 2  parts 

Place  the  films  in  this  for  i  hour  at  room  temperature  or 
heat  in  an  oven  for  5  to  15  minutes  at  55°  C.  Wash  the 
preparation  with  distilled  water,  then  with  absolute  alcohol  for 
from  3  to  4  minutes,  and  again  very  thoroughly  in  distilled 
water.     It  is  now  ready  to  treat  with  Solution  B. 

SOLUTION   B    (sensitizing   BATH) 

This  is  a  5%  solution  of  silver  nitrate  in  distilled  water. 
Allow  the  films  to  be  in  this  for  from  2  to  3  minutes.  Then 
without  washing  transfer  to  Solution  C. 

solution  c  (reducing  and  strengthening  bath) 

Gallic  acid 5  grams 

Tannin 3  grams 

Fused  potassium  acetate 10  grams 

Distilled  water 35°  cc. 

Keep  in  this  for  from  i  to  i^  minutes.  Wash,  dry,  and 
mount.  It  will  also  be  found  an  advantage  to  use  a  fresh  supply 
of  Solution  C  for  each  preparation,  a  small  quantity  being 
sufficient.  If  overbrowned,  the  background  will  be  too  deeply 
stained ;  if  underbrowned,  the  flagella  will  be  too  faint. 


STAINING  TUBERCLE   BACTERIA  6/ 

EXERCISE  XX 

STAINING  TUBERCLE  BACTERIA  (BACILLI) 

98.  The  stained  tubercle  bacteria  possess,  because  of  the 
layer  of  fatty  acids  covering  them,  the  power  of  retaining 
the  coloring  matter  even  when  treated  with  a  strong  decolor- 
izer,  such  as  a  solution  of  sulphuric  or  nitric  acid.  On  this 
account  staining  has  a  high  differential  value  which  is  made 
use  of  in  identifying  this  organism.  Thus  in  the  examination 
of  sputum  in  cases  of  suspected  tuberculosis  the  object  is  to 
determine  the  presence  of  tubercle  bacteria.  As  this  organism 
is  not  easily  cultivated,  the  staining  process  is  very  largely 
depended  upon  in  making  a  differential  diagnosis. 

99.  Work  for  this  exercise.  Make  4  cover-glass  prepara- 
tions from  a  culture  of  tubercle  (furnished).  Stain  2  of  them 
with  tubercle  stain  and  carefully  describe  the  appearance  of 
the  bacteria  and  illustrate  with  a  few  drawings. 

Stain  2  of  the  preparations  after  Gabbett's  method. 

Stain  a  cover-glass  preparation  of  tubercular  sputum  (fur- 
nished). 

For  the  next  exercise  liquefy  two  large  tubes  of  agar  and  two 
of  gelatin  and  pour  them  into  Petri  dishes.  After  the  medium 
has  soHdified  remove  the  covers  of  the  Petri  dishes  and  expose 
one  of  each  to  the  air  for  five  minutes  and  one  of  each  for 
ten  minutes.  Return  the  covers  and  place  the  agar  plates  in 
the  incubator  and  the  gelatin  ones  in  the  locker.  When  these 
plates  are  examined  in  the  next  and  subsequent  exercises  there 
will  be  a  colony  for  each  bacterium  that  fell  upon  the  medium 
from  the  air.  It  will  be  necessary  to  look  out  for  impure  or 
mixed  colonies,  as  two  or  more  organisms  may  have  fallen 
together. 

Read  the  directions  in  the  text-books  for  staining  tubercle 
bacteria  (bacilH). 


68  LABORATORY  BACTERIOLOGY 

100.  Staining  tubercle  bacteria.  Prepare  the  cover-glass 
preparations  from  the  culture  of  tubercle  bacteria  and  flame 
them  as  already  described.  Stain  in  fresh  carbol  fuchsin. 
Place  a  few  drops  of  the  stain  on  the  film  side  of  the  cover 
glass  and  hold  it  over  a  flame  with  forceps  until  steam  is  given 
off.  Allow  the  hot  stain  to  act  for  from  3  to  5  minutes,  or 
the  preparation  may  be  floated  on  the  carbol  fuchsin  in  a 
watch  glass  without  heat.  In  this  case  it  is  allowed  to  act 
for  from  10  to  15  minutes.  The  preparation  is  then  rinsed 
in  water  and  decolorized  by  treating  it  with  a  10%  solution 
of  nitric  or  sulphuric  acid  for  from  |-  to  i  minute.  It  is  again 
rinsed  in  water,  when  it  is  ready  for  examination.  It  can  be 
dried  and  mounted  permanently  in  balsam.  The  tubercle 
bacteria  should  be  stained  a  deep  reddish  color.  All  other  bac- 
teria or  animal  tissue  in  the  preparation  should  be  unstained. 
If  desired,  a  counterstain,  such  as  alkaline  methylene  blue,  may 
be  used  after  decolorizing ;  that  is,  the  preparation  should  be 
again  stained  for  about  i  minute  in  alkaHne  methylene  blue, 
rinsed  in  water,  and  examined  as  before.  In  these  prepara- 
tions the  tubercle  bacteria  are  red  and  the  other  organisms 
and  cells  are  blue.  A  counterstain  is  of  no  value  in  prepara- 
tions made  from  pure  cultures  or  for  simple  diagnostic  pur- 
poses. When  a  counterstain  is  desired  Gabbett's  decolorizing 
and  counterstaining  solution  is  very  convenient. 

gabbett's  solution 

Methylene  blue  (powder) 2  grams 

10  %  sulphuric  acid 100  cc. 

After  staining  with  the  carbol  fuchsin  treat  the  preparations 
with  this  mixture  until  the  film  has  a  faintly  bluish  tint.  This 
solution  decolorizes  and  counterstains  at  the  same  time.  This 
organism,  like  some  other  pathogenic  bacteria,  takes  the  Gram 
stain.^ 

^  See  Novy's  Laboratory  Work  in  Bacteriology,  p.  289,  for  a  list  of 
such  organisms. 


A  STUDY  OF  CERTAIN  SAPROPHYTIC  BACTERIA  69 

EXERCISE  XXI 

A  STUDY  OF  CERTAIN  SAPROPHYTIC  BACTERIA 

101.  It  is  desirable  to  have  a  definite  knowledge  concerning 
the  characters  and  properties  of  the  commonly  encountered 
species  and  groups  of  saprophytic  bacteria.  It  is  likewise 
important  to  understand  the  method  of  identifying  species. 
For  these  reasons  a  few  exercises  on  saprophytic  bacteria, 
especially  from  air,  milk,  and  water,  have  been  introduced. 

102.  Work  for  this  exercise.  Examine  and  carefully  describe 
the  cultures  made  by  exposing  agar  and  gelatin  plates  to  the 
air  in  the  last  exercise.  Determine  the  number  of  different 
colonies  and  carefully  describe  each.  Make  a  microscopic 
examination  (hanging-drop)  of  the  bacteria  in  one  of  each  of 
the  different  kinds  of  colonies  and  determine  its  genus. 

Make  for  examination  in  the  next  exercise  a  series  of  three 
plate  cultures  in  gelatin  and  one  of  two  plates  in  agar  from 
a  sample  of  milk  furnished.  The  milk  will  be  either  freshly 
drawn  in  sterile  flasks  or  samples  of  market  milk. 

103.  Identifying  species  of  bacteria.  The  genera  of  bacteria 
are  determined  by  the  morphology.  Thus  a  spherical  organ- ^ 
ism  is  a  micrococcus^  a  motile,  rod-shaped  one  is  a  bacillus^  and 
a  nonmotile,  rod-shaped  one  a  bacterium.  Each  genus  has  a 
large  number  of  species.  This  requires  some  method  by  which 
bacteria  which  look  exactly  alike  under  the  microscope  may 
be  differentiated  provided  they  are  different.  This  method 
consists  in  the  study  of  the  growth  of  these  bacteria  on  the 
different  media  and  possibly  their  effect  upon  small  animals. 
For  example,  the  B,  typhosus  and  B,  coli  comfnunis  look  so 
nearly  alike  that  one  could  not  be  sure  of  a  difference  micro- 
scopically ;  B.  coli  communis  coagulates  milk,  B,  typhosus  does 
not ;  B,  coli  communis  produces  gas  in  glucose  media,  B,  typho- 
sus does  not.     Knowing  these  properties  and  having  these 


70         LABORATORY  BACTERIOLOGY 

cultures  we  could  readily  tell  the  one  from  the  other.  To 
identify  species,  therefore,  one  must  compare  the  cultural 
characters  of  the  organism  in  question  with  the  description  of 
the  species  already  known.  As  new  media  are  constantly  being 
introduced,  one  often  finds  that  the  descriptions  given  in  text- 
books and  manuals  of  bacteriology  are  very  brief  and,  as  com- 
pared with  modern  requirements,  insufficient  to  identify  the 
species.  This  has  resulted  in  the  listing  of  a  very  large  num- 
ber of  species  that  are  very  difficult  if  not  impossible  to  iden- 
tify. With  pathogenic  bacteria  the  somewhat  specific  action 
on  experimental  animals  affords  much  aid  in  their  identifica- 
tion. For  further  explanations,  see  text-books.  Matzuschita's 
Bacteriologische  Diagnostik  is  especially  helpful  in  diagnosing 
species. 


A  STUDY  OF  BACTERIA  IN  MILK  7I 

EXERCISE   XXII 

A  STUDY  OF   BACTERIA  IN  MILK 

104.  It  is  desirable  to  understand  somewhat  clearly  the 
bacterial  contents  of  milk  and  to  know  something  of  the 
physiological  properties  of  these  bacteria.  For  this  reason 
it  is  desirable  to  study  though  but  briefly  the  bacteria  in 
ordinary  market  milk. 

References.  Russell,  Dairy  Bacteriology;  also  one  by  Conn 
and  one  by  Grotenfelt.  Hunziker,  Germicidal  action  in  cow's  milk, 
Bulletin  No.  197,  Cornell  Univ.  Exp.  Station.  Ward,  The  inva- 
sion of  the  udder  by  bacteria,  Bulletin  No.  i  y8,  Ibid.  Park,  Bac- 
terial contamination  of  the  milk  of  our  cities.  The  N.Y.  Univ. 
Bulletin  of  the  Med.  Science,  Vol.  I.  Moore,  Bacteria  in  milk. 
Report  of  the  Com.  of  Agriculture  of  N.Y.,  1902. 

105.  Work  for  this  exercise.  Examine  the  plate  cultures  made 
from  milk.  Describe  the  different  kinds  of  colonies  and  state 
approximately  the  number  of  each.  Examine  microscopically 
the  bacteria  in  one  of  each  kind  of  colony  and  determine  its 
genus. 

Inoculate  a  tube  of  milk  and  one  of  gelatin  from  each  of 
three  different  kinds  of  colonies,  stating  the  genus  of  the 
bacteria  in  each. 

Inoculate  Group  A  of  media  from  a  culture  of  B.  prodigiosus 
furnished. 

Make  for  examination  at  the  next  and  following  exercises 
three  gelatin  plates  from  a  sample  of  water  furnished  (unfil- 
tered  creek  or  well  water),  using  for  each  culture  the  quantity 
designated  by  the  instructor.  The  quantity  will  depend  upon 
the  condition  of  the  water. 


72  LABORATORY  BACTERIOLOGY 

EXERCISE    XXIII 

A  STUDY  OF  BACTERIA  IN  WATER 

106.  It  is  important  to  know  something  of  the  bacterial  con- 
tents of  water  as  it  is  found  in  wells  and  streams,  and  to  com- 
pare the  bacteria  ordinarily  found  in  water  with  those  present 
in  freshly  drawn  milk.  It  will  be  observed  that  the  normal 
bacterial  flora  of  water  and  of  milk  are  quite  different. 

References.  Percy  and  G.  C.  Frankland,  Micro-organisms 
in  Water.  Clark  and  Gage,  34th  Annual  Report  Massachusetts 
State  Board  of  Health.  Jordan,  The  kinds  of  bacteria  found 
in  river  water,  The  Journal  of  Hygiene,  Vol.  Ill,  No.  i  (1903). 

107.  Work  for  this  exercise.  Carefully  examine  and  describe 
the  cultures  made  from  the  colonies  on  the  milk  plates  in  the 
last  exercise. 

Examine  and  describe  the  colonies  on  the  plate  cultures 
made  from  water.  Determine  the  number  of  colonies  and 
approximately  the  number  of  each  kind  of  colony  on  the 
plate. 

Examine  microscopically  the  bacteria  from  one  of  each 
kind  of  colony  and  determine  the  genus. 

Inoculate  a  tube  of  milk  and  one  of  gelatin  from  each  of 
three  different  colonies,  if  there  are  as  many.  In  later  exer- 
cises examine  these  cultures  and  compare  them  with  those 
made  from  colonies  of  milk  bacteria. 

Examine  and  describe  the  cultures  of  B.  prodigiosiis. 

Inoculate  groups  of  media  A  and  B  from  a  culture  of  Ps, 
fluorescens  liquefaciens  furnished. 


A  STUDY  OF  CERTAIN  PYOGENIC  BACTERIA    73 

EXERCISE    XXIV 

A  STUDY  OF  CERTAIN  PYOGENIC  BACTERIA 

108.  There  are  a  number  of  bacteria  which  are  able  to 
cause  suppuration,  but  ordinarily  the  formation  of  pus  is  due 
to  the  presence  of  certain  streptococci  and  micrococci.  A 
number  of  bacilli,  especially  B,  coli  commtmis  and  Fs.  pyocy- 
aneus^  are  frequently  found  as  the  apparent  cause  of  suppura- 
tion. As  it  is  impossible  to  study  more  than  a  very  few  of  these 
species,  two  of  the  most  common  and  one  of  the  more  rarely 
encountered  organisms  in  suppurating  wounds  and  abscesses 
are  chosen  for  special  study. 

References.  Chapters  on  pyogenic  bacteria  in  text  and  refer- 
ence books.  Christman  (recherches  sur  la  suppuration),  Annals 
de  rinst.  Pasteur,  Vol.  II  (1888),  p.  469.  Lucet  (in  animals),  Ibid., 
Vol.  VII  (1893),  p.  325.  Von  Lingelsheim  (concerning  strepto- 
cocci), Zeitschrif t  fiir  Hygiene,  Bd.  X  (1892),  S.  331.  Moore,  Am. 
Vet.  Review,  January,  February,  and  March,  1900. 

109.  Work  for  this  exercise.  Inoculate  a  tube  of  each 
medium  in  Groups  A  and  B  from  each  of  the  cultures  of 
Streptococcus  pyogenics  and  Micrococcus  pyogenes  aureus  which 
will  be  furnished. 

Describe  the  cultures  of  Fs,  fl-uorescens  liquefaciens  and  ex- 
amine the  bouillon  culture  microscopically  in  a  hanging-drop 
and  in  a  stained  preparation. 

Examine  the  milk  and  gelatin  cultures  made  from  the 
colonies  from  milk  and  water  plates. 

Read  carefully  the  chapter  on  pyogenic  bacteria  in  the 
text-books. 

Give  in  the  notes  a  definition  of  each  of  the  following  terms  : 
saprophytic,  parasitic,  pathogenic,  and  pyogenic  bacteria.  See 
text-books. 


74  LABORATORY   BACTERIOLOGY 

EXERCISE    XXV 

PYOGENIC    BACTERIA  {continued) 

110.  Work  for  this  exercise.  Examine  and  carefully  describe 
the  cultures  made  in  Exercise  XXIV.  Note  especially  the 
growth  on  the  agar,  gelatin,  and  potato,  and  in  the  tubes  of 
the  bouillon  containing  the  sugars.  In  describing  the  color 
use  color  charts  which  are  in  the  laboratory. 

Examine  microscopically  in  (i)  hanging-drop  and  (2)  stained 
cover-glass  preparations  the  bacteria  from  the  bouillon  and 
agar  cultures. 

Measure  a  few  of  the  bacteria  in  the  stained  preparations 
from  the  agar  cultures,  and  make  a  drawing  of  them,  magnified 
1000  diameters. 

Inoculate  for  Exercise  XXVI  a  tube  of  each  medium  in 
Groups  A  and  B  from  a  culture  (furnished)  of  Ps,  (Bacillus) 
pyocyaneus. 

For  suggestions  in  studying  cultures  and  microscopic  prep- 
arations of  bacteria,  see  Exercises  VI  and  XII. 

Include  in  the  notes  the  names  of  the  different  forms  of 
Micrococcus  pyogenes  and  a  classification  of  streptococci. 


PSEUDOMONAS   PYOCYANEUS  75 

EXERCISE    XXVI 

PSEUDOMONAS    PYOCYANEUS 

111.  Fseudo7iionas  pyocyaneus^  commonly  known  as  the 
bacillus  of  green  pus,  blue  pus,  or  blue-green  pus,  is  quite 
widely  distributed  in  nature.  While  ordinarily  it  has  been 
considered  of  little  pathogenic  importance,  it  is  known  to  pos- 
sess at  times,  and  under  certain  conditions,  marked  infecting 
powers.  This  organism  has  been  called  the  honey  bacillus, 
on  account  of  the  peculiar  odor  emitted  from  its  cultures.  It 
is  to  be  differentiated  from  Ps.  fitiorescens  liquefaciens  and  its 
varieties  which  frequently  appear  in  water. 

References.  Chapters  on  this  organism  in  text-books. 
Barker,  The  clinical  symptoms,  etc.,  The  Jour,  of  the  Am.  Med. 
Asso.,  July  31,  1897.  Lartigau,  Study  of  pathogenesis,  Jour,  of 
Exp.  Med.,  1898,  p.  595.  Jordan,  Pigments  produced  by.  Ibid., 
1899,  p.  627.  Ruzicka,  Arch,  fiir  Hygiene,  Bd.  XXXIV,  S.  149, 
andBd.  XXXVII,  S.  i. 

112.  Work  for  this  exercise.  Examine  very  carefully  and 
describe  fully  the  cultures  of  Ps.  pyocyaneus  made  during  the 
last  exercise. 

Make  and  examine  a  hanging-drop  and  a  stained  cover- 
glass  preparation  from  each  of  the  bouillon  and  agar  cultures. 

Describe  the  appearance  of  the  bacteria  in  each. 

Measure  and  make  a  drawing  of  a  few  organisms  in  the 
preparation  from  the  agar  culture.     Magnify  500  diameters. 

Reexamine  the  cultures  of  the  streptococcus  and  the  micro- 
coccus studied  in  the  last  exercise  and  note  all  appreciable 
changes  which  have  taken  place. 

Inoculate  a  tube  of  each  of  the  media  in  Groups  A  and  D 
from  a  culture  of  B,  coli  comfnunis  (furnished)  for  study  at 
the  next  exercise. 


'jG  LABORATORY  BACTERIOLOGY 

EXERCISE    XXVII 

BACILLUS  COLI  COMMUNIS 

113.  Of  the  bacteria  normally  present  on  the  mucous  mem- 
branes of  the  animal  body  the  colon  group  is,  on  account  of 
its  close  morphological  relationship  to  the  bacilli  of  typhoid 
fever  and  hog  cholera,  of  more  than  ordinary  interest.  There 
are  varieties  of  this  organism  which  approximate  very  closely 
in  their  biochemic  properties  as  well  as  in  their  morphology  to 
the  typhoid  and  also  to  the  hog-cholera  bacilli.  It  is  impor- 
tant that  this  existing  variation  be  recognized  and  that  the 
list  of  properties  which  characterize  B.  coli  communis  should 
be  clearly  determined.  The  differentiation  of  the  colon  and 
typhoid  bacilli  as  they  exist  in  nature  is  one  of  the  diffi- 
cult problems  in  practical  bacteriological  work.  The  culture 
assigned  approaches  very  closely  to  the  typical  species. 

References.  Chapters  on  this  organism  in  the  text-books. 
T.  Smith,  The  Am.  Jour,  of  Med.  Sci.,  September,  1896.  Adelaide 
W.  Peckham,  Jour,  of  Exp.  Med.,  Vol.  II  (1897),  p.  549.  Adami, 
Ibid.,  Vol.  IV  (1899),  p.  349.  Gage  and  Phelps,  Report  Am. 
Pub.  Health  Asso.,  1902,  p.  402.  Moore  and  Wright,  Am.  Med., 
Vol.  Ill  (1902),  p.  504. 

114.  Work  for  this  exercise.  Describe  the  appearance  of 
each  of  the  cultures  of  B,  coli  co?nmu?iis  made  in  Exercise 
XXVI. 

Examine  the  bacteria  in  a  hanging-drop  preparation  from 
the  bouillon  and  glucose  bouillon  cultures. 

Make  and  stain  with  carbol  fuchsin  a  cover-glass  preparation 
from  the  agar  culture.  Measure  a  few  of  the  bacilli  and 
record  their  size  in  the  notes. 

Note  especially  the  quantity  of  gas  formed  in  each  of  the 
fermentation  tubes.     These  cultures  should  be  kept  until  the 


BACILLUS   COLl   COMMUNIS  7/ 

next  exercise,  when  they  should  be  examined  again.  If  the  gas 
formation  is  then  completed,  determine  the  quantity  in  each 
tube  and  the  ratio  of  CO2  to  H. 

Make  two  gelatin  plates  from  the  bouillon  culture.  In  mak- 
ing these  plates  use  a  tube  of  sterihzed  distilled  water  for  the 
first  dilution. 

Inoculate  Group  D  of  media  with  the  paracolon  bacillus 
from  a  culture  furnished. 

Test  the  culture  in  sugar-free  bouillon  for  the  presence  of 
indol  if  it  is  at  least  72  hours  old.  Read  the  chapter  on  this 
organism  in  the  text-books. 

115.  The  indol  (cholera-red)  test.  Add  i  cc.  of  a  .01  %  solu- 
tion (fresh)  of  potassium  nitrite  and  a  few  drops  of  concen- 
trated sulphuric  acid  to  the  culture  in  sugar-free  bouillon. 
A  pinkish  color  indicates  the  presence  of  indol.  In  an  old 
(3  to  5  day)  culture  the  reaction  is  usually  stronger  than  in 
a  more  recently  made  one. 

If  sugar-free  bouillon  is  not  at  hand,  a  tube  of  Dunham's 
solution  can  be  used  instead  with  quite  good  results. 

J^ifig  method.  When  there  is  a  small  quantity  of  indol  it 
can  be  detected  more  readily  by  the  "ring  method."  Add  i 
or  2  cc.  of  a  2  5  %  solution  of  H2SO4,  allowing  it  to  run  down 
on  the  inside  of  the  tube  containing  the  culture.  Add  i  cc. 
of  potassium  nitrite.  If  indol  is  present,  a  pinkish  ring  will  be 
observed  between  the  layer  of  acid  in  the  bottom  of  the  tube 
and  the  culture  above  it. 

116.  Dunham's  peptone  solution.  This  is  simply  a  solution 
of  peptone  and  sodium  chloride  in  distilled  water.  The  for- 
mula is  as  follows  : 

Dried  peptone i    gram 

.    Sodium  chloride 0.5  gram 

Distilled  water 100    cc. 

Dissolve  the  peptone  and  salt  in  the  water,  distribute  it 
in  the  tubes  (5  cc.  each),  and  sterilize  the  same  as  bouillon. 


7^  LABORATORY   BACTERIOLOGY 

EXERCISE   XXVIII 

BACILLUS  COLI  COMMUNIS  AND  THE  PARACOLON 

117.  Work  for  this  exercise.  Reexamine  the  cultures  of 
B.  coH  communis  and  note  any  changes  which  have  occurred 
in  their  appearance.  Determine  the  gas  formula  in  the  fer- 
mentation tubes  with  the  different  sugars.  Place  the  milk  and 
litmus-milk  cultures  in  the  incubator  and  examine  them  later. 

Examine  carefully  and  describe  the  cultures  of  the  paracolon 
bacillus.  Examine  microscopically  at  least  one  culture.  Note 
especially  its  motiUty. 

Examine  and  describe  fully  the  colonies  on  the  gelatin  plates. 
Preserve  the  plates  and  examine  them  at  the  following  exercises. 

Examine  microscopically,  in  a  stained  preparation,  the  bac- 
teria from  a  colony  on  the  gelatin  plate.  Preserve  a  prepara- 
tion to  accompany  the  notes. 

Isolate  B,  coli  communis  from  the  intestine  of  an  animal. 
The  intestine  will  be  furnished. 

Inoculate  with  B,  cholerce  suis,  for  Exercise  XXIX,  a  tube 
of  each  medium  in  Groups  A  and  D,  and  similar  tubes  with 
B,  typhosus^  from  the  cultures  furnished. 

118.  Isolating  B.  coli  communis  from  the  intestine.  Care- 
fully open  the  intestine  by  a  longitudinal  incision.  Scrape 
away  the  contents,  if  any,  from  a  small  area  of  the  mucous 
membrane.  Take  a  loopful  of  the  mucus  from  the  surface  of 
the  mucous  membrane  and  inoculate  a  large  tube  of  Hquefied 
gelatin  with  it.  After  shaking  the  tube  carefully,  inoculate  a 
second  tube  with  2  loopfuls  from  the  first,  and  a  third  with  3 
loopfuls  from  the  second.  Pour  the  gelatin  into  Petri  dishes 
and  label  them.  These  plates  should  be  examined  daily. 
The  colonies  of  B,  coli  communis  can  be  distinguished  from 
others  which  may  appear  by  their  thin  spreading  growth, 
sharply  defined  but  irregular  borders,  and  their  bluish  appear- 
ance, especially  with  transmitted  light.  Compare  with  colo- 
nies on  gelatin  plates  from  a  pure  culture  (Exercise  XXVII). 


BACILLUS  CHOLER.E   SUIS  79 

EXERCISE   XXIX 

BACILLUS  CHOLERA   SUIS  AND   BACILLUS  TYPHOSUS 

119.  The  bacilli  of  typhoid  fever  and  of  hog  cholera  resem- 
ble each  other  very  closely  morphologically  and  in  certain 
of  their  cultural  characters  and  biochemic  properties.  Like 
B.  coli  communis  each  of  these  organisms  has  several  varieties. 
Already  several  distinct  varieties  of  the  hog-cholera  bacillus 
have  been  described.-^ 

Certain  of  the  varieties  of  these  species  approach  each  other 
very  closely,  while  others  approach  B,  coli  communis  in  their 
various  manifestations.  It  is  important,  therefore,  that  the 
morphology  and  properties  of  each  of  these  species  should  be 
carefully  determined.  The  fact  should  be  kept  clearly  in  mind 
that  while  these  two  species  and  the  colon  bacillus  resemble 
each  other  in  certain  directions,  they  are,  so  far  as  has  yet 
been  demonstrated,  distinct  species.  The  sp.ecial  methods  of 
differentiation  must  be  omitted  from  this  elementary  course. 
Read  carefully  the  chapter  on  B,  typhosus  in  the  text-book. 

References.  To  hog  cholera.  Salmon,  Special  Report  on  Hog 
Cholera,  Bureau  of  Animal  Industry,  U.  S.  Depart,  of  Agric,  1889. 
Smith,  Bulletin  No.  6,  Ibid.,  1894.  Moore,  Report  N.  Y.  State 
Commissioner  of  Agriculture,  1897.  The  Am.  Vet.  Review,  March, 
1898. 

References.  To  typhoid.  Chapters  on  this  organism  in  text- 
books. Eberth,  Virchow's  Archiv,  Bd.  81,  1880.  Ibid.,  Bd. 
83,  1 88 1.  Gaffky,  Mittheilungen  aus  d.  Kais.  Gesundheitsamte, 
Vol.  II,  1884,  S.  372.  Losener,  Arbeiten  aus  d.  Kais.  Gesund- 
heitsamte, Vol.  XI,  1895.  P^r^,  Annales  de  ITnst.  Pasteur,  Vol.  VI, 
1882,  p.  512.    Jordan,  Medical  News,  September  28,  1895.   Flexner, 

1  The  hog-cholera  group  of  bacteria,  Bulletin  No.  6,  U.  S.  Bureau  of 
Animal  Industry,  p.  9. 


So  LABORATORY  BACTERIOLOGY 

The  Johns  Hop.  Hosp.  Reports,  Vol.  V,  p.  343.  Smith,  The  Jour, 
of  the  Boston  Soc.  of  Med.  Sciences,  June,  1898.  Hiss,  The 
Jour,  of  Exp.  Med.,  Vol.  II,  1887,  p.  677. 

120.  Work  for  this  exercise.  Examine  the  plate  cultures 
made  from  the  intestine  for  the  colon  bacillus. 

Determine  the  approximate  number  of  colonies  on  each 
plate  and  note  especially  the  number  of  colonies  of  jB,  colt 
communis  and  describe  their  appearance. 

Inoculate  a  'tube  of  agar,  one  of  milk,  and  a  fermentation 
tube  of  glucose  bouillon  from  one  of  the  colonies.  Study 
these  cultures  in  the  next  exercise  and  compare  them  with 
the  notes  on  cultures  of  B.  coli  communis  in  these  media. 

Examine  and  carefully  describe  the  cultures  of  B.  cholerce 
suis  and  B,  typhosus.  Note  especially  the  reaction  of  the  cul- 
tures in  the  fermentation  tubes.  Examine  the  bouillon  cul- 
tures microscopically  in  hanging-drop  and  in  stained  cover- 
glass  preparations.     Describe  the  appearance  of  the  bacteria. 

Make  a  series  of  3  gelatin  plate  cultures  from  the  bouillon 
culture  of  each  organism. 


BACILLUS   CHOLERA   SUIS  8l 


EXERCISE   XXX 

BACILLUS  CHOLERA  SUIS  AND   BACILLUS   TYPHOSUS 

{conti7iued^ 

121.  Work  for  this  exercise.  Reexamine  all  of  the  cultures 
of  B,  cholercB  siiis  and  B.  typhosus.  Note  especially  the  con- 
dition of  the  fermentation  tubes.  Keep  the  milk  and  the  lit- 
mus-milk cultures  in  the  incubator  for  about  5  weeks  and  note 
any  changes  which  may  take  place  from  week  to  week. 

Examine  and  carefully  describe  the  colonies  on  the  gelatin 
plates. 

Try  the  indol  test  (§  115)  with  the  culture  in  sugar- free 
bouillon. 

Make  and  stain  with  alkahne  methylene  blue  a  few  (3  or  4) 
cover-glass  preparations  from  the  organs  (liver,  spleen,  kidney, 
or  blood)  of  a  rabbit  which  has  died  from  the  effects  of  the 
inoculation  with  hog-cholera  bacilli.  Note  the  number  (few 
or  many)  of  bacteria  in  the  preparations  and  preserve  one 
of  them  to  accompany  the  notes.  Make  a  drawing  of  a  few 
bacilli. 

Examine  and  complete  the  notes  on  the  culture  of  B,  coli 
communis  and  the  paracolon  bacillus.  Compare  them  with 
the  cultures  of  hog-cholera  and  typhoid  bacteria. 

122.  Making  cover-glass  preparations  from  tissues.  With  a 
pair  of  fine  forceps  take  up  a  bit  of  tissue  from  the  freshly  cut 
liver,  spleen,  or  kidney,  and  rub  it  gently  over  the  surface  of 
a  clean  cover  glass,  care  being  taken  that  the  film  of  tissue 
is  thin.  Allow  this  to  dry  in  the  air,  after  which  pass  the 
cover  glass,  film  up,  three  times  through  the  flame  to  fix  the 
tissue  to  the  glass.  It  can  be  stained  the  same  as  the  cover- 
glass  preparations  from  the  cultures.  When  carbol  fuchsin 
is  used  for  staining,  the  preparation  should  be  wet  before 
applying.     These  are  often  spoken  of  as  smear  preparations. 


82  LABORATORY  BACTERIOLOGY 

In  making  these  preparations  from  blood,  hold  a  cover  glass 
by  the  edge  with  a  pair  of  dissecting  forceps.  With  the  plati- 
num loop  place  a  drop  of  blood  on  the  cover  glass  near  the 
forceps.  Take  a  thick,  square  cover  glass  by  the  edge,  rest  it 
on  the  first  above  the  drop  of  blood,  hold  it  at  an  angle  of 
about  20°  from  it,  and  draw  it  down  over  the  first,  thus  spread- 
ing the  blood  in  a  very  thin,  even  film  over  the  surface.  If 
the  film  is  thick,  the  preparation  should  be  rejected  and 
another  made. 


BACILLUS   CHOLERA   SUIS  83 


EXERCISE  XXXI 

BACILLUS  CHOLERA   SUIS  AND   BACILLUS  TYPHOSUS 

{continMed) 

123.  Work  for  this  exercise.  Reexamine  and  complete  the 
notes  on  all  of  the  cultures  except  the  milk  and  litmus  milk. 
Carefully  observe  the  reaction  of  all  the  liquid  cultures. 

Compare  the  colonies  on  the  gelatin  plates  with  those  of 
B,  coli  communis. 

Make  a  careful  comparison,  in  tabulated  form,  of  the  mor- 
phology, including  measurements,  of  the  bacilli  themselves 
and  of  the  appearance  of  the  growth  in  the  different  cultures 
of  B,  coli  communis  and  the  bacilli  of  hog  cholera  and  typhoid 
fever.     Add  the  cultural  characters  of  the  paracolon  bacillus. 

The  cultures,  excepting  those  in  milk,  can  be  rejected  now, 
or,  if  desired,  they  may  be  kept  for  further  study  and  com- 
parison. Inoculate  the  media  in  Groups  A  and  B  with  the 
bacillus  of  dysentery  from  a  culture  furnished. 


84  LABORATORY  BACTERIOLOGY 

EXERCISE   XXXII 

BACILLI  OF  DYSENTERY 

124.  A  number  of  bacilli  have  been  isolated  from  the  intes- 
tine from  cases  of  dysentery.  The  first  of  these  organisms 
seems  to  have  been  described  by  Shiga,  who  isolated  it  from 
cases  of  dysentery  in  India.  Since  his  work  was  published  a 
number  of  bacilli  very  closely  related  to  the  one  he  described, 
if  not  identical  with  it,  have  been  found  in  this  country.  In 
this  exercise  one  or  more  of  these  organisms  will  be  studied. 

References.  Shiga,  Centralblatt  F.  Bakt.,  Vol.  XXIII,  p.  599, 
Deutsche  Med.  Wochensch.,  Vol.  XXVII,  p.  741.  Kruse,  Ibid., 
p.  370.  Duval  and  Bassett,  Am.  Med.,  Vol.  IV,  p.  417.  Gay, 
Univ.  Penn.  Med.  Bulletin,  Vol.  XV,  p.  307.  Duval  and  Gay, 
Ibid.,  Vol.  XVI,  1903,  p.  177.  Flexner,  Ibid.,  Vol.  XIV,  p.  190. 
Park  and  Carey,  Jour.  Med.  Research,  Vol.  IX,  1903,  p.  180. 

125.  Work  for  this  exercise.  Study  and  carefully  describe 
the  cultures  of  dysentery  bacteria  made  in  the  last  exercise. 

Examine  the  bacteria  in  the  hanging-drop  and  in  stained 
cover-glass  preparations  from  the  bouillon  and  gelatin  cultures. 
Describe  the  morphology  and  make  a  drawing  of  a  few  bacilli, 
giving  their  size  by  actual  measurement. 

Compare  carefully  the  cultural  characters  of  this  organism 
with  those  of  hog-cholera,  typhoid,  and  colon  bacilli. 


WIDAL  SERUM   TEST  85 

EXERCISE  XXXIII 

WIDAL  SERUM  TEST 

126.  This  test  depends  upon  the  fact  that  when  the  blood 
serum  of  a  person  suffering  with  typhoid  fever,  or  who  has 
recently  recovered  from  it,  is  added  to  a  bouillon  culture  of 
the  bacillus,  the  bacilli  become  less  motile  and  soon  aggluti- 
nate in  small  clumps.  The  dilutions  used  vary  from  equal  parts 
of  serum  and  culture  to  dilutions  of  i  to  50,000.  It  is  recom- 
mended that  the  stronger  dilutions  shall  be  used,  i.e.  those 
from  I  :  10  to  i  :  50.  The  test  has  proven  to  be  of  much 
diagnostic  value  in  typhoid  fever. 

It  has  been  found  that  a  similar  reaction  will  take  place 
with  certain  other  bacteria  when  they  are  brought  in  contact 
with  the  serum  from  animals  suffering  from  the  disease  which 
they  produce.  Thus  it  has  been  shown  that  such  a  reaction 
occurs  with  hog-cholera  bacilli  and  serum  from  affected  or 
immunized  animals. 

On  account  of  the  diagnostic  value  of  this  reaction  it  is 
employed  very  extensively  in  many  health  departments  for 
the  diagnosis  of  typhoid  fever. 

References.    Black,  Johns  Hop.  Hosp.  Bulletin,  December, 

1896.  Welch,   The  Jour,  of  the  Am.   Med.  Assc,   August   14, 

1897.  Johnston,  N.Y.  Med.  Jour.,  October  31,  1896;  Med.  News, 
January  23,  1896.  Biggs  and  Park,  The  Am.  Jour,  of  the  Med. 
Sci.,  March,  1897.  Wesbrook  and  Wilson,  The  Phila.  Med.  Jour., 
March  26,  1898.  Dawson,  N.Y.  Med.  Jour.,  February  20,  1897 
(concerning  hog  cholera).  Cabot,  Serum  Diagnosis  of  Disease, 
1899.  Ruediger,  The  Jour,  of  Infect.  Diseases,  Vol.  I,  p.  236. 
See  also  recent  text-books. 

127.  Work  for  this  exercise.  Take  one  loopful  of  a  fresh 
bouillon  culture  of  typhoid  baciUi  (which  will  be  furnished)  and 
place  it  on  a  cover  glass,  add  one  loopful  of  diluted  blood 


86         LABORATORY  BACTERIOLOGY 

serum  from  a  typhoid  patient,  or  the  blood  of  an  immune 
guinea  pig,  and  immediately  make  a  hanging-drop  prepara- 
tion with  a  loopful  of  the  mixture  and  examine.  Note  the 
effect  on  the  motility  of  the  bacilH  and  their  aggregation  into 
clumps.  Specify  the  time  elapsing  before  the  agglutination 
appears  and  the  time  required  for  the  complete  clumping. 

Make  a  similar  examination  of  a  culture  to  which  i/io 
blood  serum  has  been  added. 

Repeat  the  above  test  with  the  blood  from  animals  affected 
with  or  immunized  against  hog  cholera. 

Examine  a  dried  specimen  of  blood  for  this  reaction.  Add 
a  few  drops  of  bouillon  or  water  to  the  drop  of  dried  blood  on 
a  shde,  and  after  it  has  become  well  mixed  add  a  loopful  of  it 
to  a  similar  quantity  of  a  fresh  bouillon  culture  and  examine 
it  immediately  in  a  hanging  drop. 

Inoculate  the  media  in  Groups  A  and  B  with  the  culture 
iirnished  of  B,  septiccemice  hemorrhagicce,  or  M,  lanceolatus^ 
or  both,  as  directed. 

128.  Securing  blood  for  the  widal  test,  {a)  Preparation  of 
dried  blood.  Prick  the  finger  or  lobe  of  the  ear  (if  a  lower 
animal,  the  shaved  ear  is  a  good  place)  sufficiently  deep  to 
procure  a  drop  of  blood.  Place  it  on  a  slide  by  means  of  a 
platinum  loop  and  allow  it  to  dry. 

{h)  Fresh  blood.  Procure  a  drop  of  blood  as  in  {a)  ;  add 
to  it  lo  drops  of  water  on  a  glass  slide  or  in  a  small  test  tube. 
Stir  until  the  blood  is  dissolved.  One  loopful  of  this  mixed 
with  a  similar  quantity  of  the  bouillon  will  give  a  dilution  of 
I  to  20. 

{c)  Serum,  From  a  similar  but  deeper  prick,  or  by  draw- 
ing a  few  drops  of  blood  from  a  vein  with  a  hypodermic  syringe, 
secure  a  few  drops  of  blood.  Place  them  in  the  bottom  of  a 
small,  short,  sterile  tube  and  allow  the  serum  to  ooze  out. 
This  can  often  be  helped  by  separating  the  blood  from  the 
tube  by  means  of  a  sterile  wire.  If  retained  for  any  length 
of  time  before  making  the  test,  the  serum  must  be  kept  in  a 


WIDAL  SERUM   TEST  ^J 

cool  place.  Experimentally,  it  is  easily  obtained  by  immu- 
nizing a  guinea  pig  and  then  drawing  the  desired  amount  of 
blood  from  a  vein. 

Blood  serum  may  be  obtained  by  filling  a  small  (capillary) 
glass  tube  with  the  fresh  blood,  sealing  it,  and  allowing  it  to 
stand  until  the  serum  collects  on  the  top,  when  the  tube  may 
be  broken  and  the  serum  drawn  off  with  a  fine-pointed  pipette. 


SS  LABORATORY  BACTERIOLOGY 


EXERCISE   XXXIV 

BACTERIUM  SEPTIC^MI^E  HEMORRHAGICE  OR 
MICROCOCCUS   LANCEOLATUS 

129.  The  first  of  these  organisms  is  the  cause  of  swine 
plague  and  a  number  of  different  diseases  in  animals,  while  the 
latter  is  the  cause  of  lobar  pneumonia  in  man. 

The  name  Bacillus  septiccemics  hemorrhagicce  was  given  by 
Hiippe  to  the  bacillus  of  swine  plague  (Smith).  This  bacte- 
rium (bacillus)  is  morphologically  and  in  its  cultural  characters 
not  distinguishable  from  the  bacterium  (bacillus)  of  rabbit 
septicaemia  (Koch),  of  fowl  cholera  (Pasteur),  and  of  Schweine- 
seiiche  (Schlitz).  It  is  similar  to  a  species  of  pathogenic  bac- 
teria found  more  or  less  frequently  in  the  upper  air  passages 
of  nearly  all  of  the  domesticated  animals.  It  is  very  similar 
also  to  a  pathogenic  bacillus  found  in  broncho  pneumonia  in 
cattle  and  an  infectious  pneumonia  in  sheep.  These  organ- 
isms are  also  known  as  the  Fasteurellose,  i.e.  belonging  to  the 
genus  Pasteurella  of  Trevison. 

Micrococcus  lanceolatus^  is  the  specific  organism  of  lobar 
pneumonia  in  man.  It  is  found  in  the  pneumonic  lung  tissue 
and  also  in  the  saliva  of  a  certain  number  of  healthy  people. 
In  many  of  its  properties  this  organism  resembles  very 
closely  the  bacterium  of  swine  plague.  In  studying  the  two 
species  together  there  will  be  good  opportunity  of  comparing 
them  and  detecting  the  differences  and  similarities  existing 
between  them. 

References.  To  swine  plague.  Smith,  Report  on  swine 
plague,  Bureau  of  Animal  Industry,  U.  S.  Depart,  of  Agric,  1891. 
Smith,  Zeitschrift  fiir  Hygiene,  Bd.  X,  1891,  S.  480.     Smith  and 

1  For  the  history  and  synonymy  of  this  organism,  see  article  by  Pro- 
fessor Welch  in  the  Johns  Hopkins  Hospital  Bulletin,  Vol.  Ill,  p.  125. 


BACTERIUM   SEPTIC^MI.^   HEMORRHAGICA        89 

Moore,  Bulletin  No.  6,  Bureau  of  Animal  Industry,  1894.  Moore, 
Report  N.Y.  State  Com.  of  Agric,  1897.  Nocard,  Am.  Vet.  Review. 
References.  To  M.  lanceolatus.  Chapters  on  Micrococcus 
lanceolatus  {dip lo coccus  pneumonicB)  in  text-books.  Welch,  Johns 
Hop.  Hosp.  Bulletin,  Vol.  Ill,  1892,  p.  125. 

130.  Work  for  this  exercise.  Carefully  examine  and  describe 
the  cultures  made  in  Exercise  XXXIII. 

Examine  the  agar  and  bouillon  cultures  microscopically  in 
both  the  living  condition  and  in  stained  cover-glass  prepara- 
tions. 

Describe  the  appearance  of  the  bacteria  and  make  a  draw- 
ing of  a  few  of  them  from  one  preparation  magnified  1000 
diameters. 

Preserve  a  preparation  to  accompany  notes. 

If  there  is  any  growth  in  the  gelatin  tube,  make  a  series  of 
3  gelatin  plates  from  the  bouillon  culture. 

Measure  a  few  of  the  bacteria  with  the  filar  micrometer  and 
record  the  measurements. 

Inoculate  a  tube  of  glycerin  agar,  ^gg  medium,  and  glycerin 
bouillon  from  a  culture  of  avian  tubercle  bacteria  for  study 
in  Exercise  XXXVI. 


go  LABORATORY  BACTERIOLOGY 


EXERCISE    XXXV 

BACTERIUM  SEPTICEMI^E   HEMORRHAGICA  OR 
MICROCOCCUS    LANCEOLATUS    {continued) 

131.  Work  for  this  exercise.  Reexamine  all  the  cultures 
of  these  bacteria,  paying  special  attention  to  the  reactions  of 
the  liquid  cultures. 

Make  the  indol  test  with  the  cultures  in  sugar-free  bouillon. 

Make,  stain,  and  examine  a  cover-glass  preparation  from  an 
organ  and  the  blood  of  a  rabbit  which  has  died  from  the  inocu- 
lation with  swine-plague  bacteria  or  with  Micrococcus  lanceo- 
latus  (the  rabbit  will  be  furnished  by  the  instructor).  Stain 
the  preparations  with  an  aqueous  solution  of  fuchsin.  Study 
the  bacteria  in  these  preparations  and  carefully  compare  the 
two.     Indicate  in  the  notes  the  differences,  if  any  are  found. 

Give  in  the  notes  a  list  of  characters  and  cultural  properties 
of  Bad,  septiccemice  hemorrhagicce  and  of  M,  lanceolatus  that 
are  of  differential  value.  In  what  properties  do  they  differ 
from  B,  cholercB  suis  and  B,  typhosus. 

Keep  the  cultures  until  the  next  exercise  and  compare  them 
again,  after  which  they  may  be  rejected. 


BACTERIUM  TUBERCULOSIS  9I 

EXERCISE    XXXVI 

BACTERIUM   TUBERCULOSIS 

132.  The  tubercle  bacterium  does  not  grow  readily  on  the 
ordinary  media.  For  its  cultivation  blood  serum,  glycerin  agar, 
or  bouillon  containing  from  5  to  7%  glycerin  is  ordinarily 
used.  Formerly  it  was  with  much  difficulty  that  it  was  made 
to  grow  from  lesions  in  tuberculous  animals  ;  but  when  a  culture 
was  once  started  it  could,  on  the  media  mentioned  above,  and 
sometimes  on  agar,  be  cultivated  in  subcultures  with  compara- 
tive ease.  More  recently  Dr.  Theobald  Smith  has  described 
a  method  which  renders  its  cultivation  from  tuberculous  lesions 
much  easier  (for  details,  see  Appendix  IV).  It  grows  very 
slowly  and  it  is  necessary  that  the  temperature  should  be  kept, 
without  variation,  at  about  37°  C.  The  avian  variety  grows 
much  more  readily  on  glycerin  agar,  egg  medium,  serum,  and 
in  glycerin  bouillon.  On  account  of  these  difficulties  it  is 
not  practicable,  in  a  general  course,  to  cultivate  this  organism, 
but  cultures  on  solid  and  liquid  media  will  be  furnished  by 
the  instructor  for  examination.  It  is  important,  however,  to 
be  able  to  recognize  this  organism  in  tissues  and  sputum,  and 
consequently  the  following  additional  exercise  in  staining  and 
studying  it  is  given. 

References.  Chapters  on  this  organism  in  text-books.  Smith, 
Jour,  of  Exp.  Med.,  1898,  p.  451.  Moore,  Med.  News,  May  14, 
1892  (methods  of  staining).  Nuttall,  Johns  Hop.  Hosp.  Bulletin, 
1 891,  Vol.  II,  p.  67.  Dorset,  Report  Am.  Public  Health  Asso., 
Vol.  XXIV,  p.  157. 

133.  Work  for  this  exercise.  Examine  and  carefully 
describe  the  appearance  of  the  cultures  of  the  tubercle 
bacterium  (human  or  bovine  variety)  on  glycerin  agar  and  in 
glycerin  bouillon  furnished. 


92         LABORATORY  BACTERIOLOGY 

Make  2  cover-glass  preparations  from  the  cultures  furnished 
for  that  purpose  and  stain  them  with  carbol  fuchsin  (§  100). 

Make  4  cover-glass  preparations  from  tuberculous  sputum 
and  stain  for  tubercle  bacteria.  It  is  often  desirable  to  counter- 
stain  the  specimens  from  sputa.  Stain  2  of  them  by  Gabbett's 
method  and  2  with  carbol  fuchsin,  and  decolorize  without 
counterstaining.  Make  a  few  (2  or  3)  cover- glass  prepara- 
tions from  the  liver  or  spleen  of  a  guinea  pig  which  has  died 
from  tuberculosis,  and  stain  them  for  tubercle  bacteria.  Stain 
one  with  carbol  fuchsin  and  decolorize  with  sulphuric  acid,  and 
stain  one  by  Gabbett's  method. 

Examine  the  cultures  of  avian  tubercle  bacteria  and  describe 
their  appearance.  Stain  and  examine  a  few  preparations  from 
one  of  the  cultures. 

Indicate  in  the  notes  the  essential  differences  between  human, 
bovine,  and  avian  tubercle  bacteria  respecting  (i)  morphology, 
(2)  cultural  properties,  and  (3)  pathogenesis. 

Measure  the  tubercle  bacteria  in  one  of  the  preparations  and 
make  a  drawing  showing  a  few  of  them  magnified  1000  diameters. 

134.  Making  cover-glass  preparations  from  sputum.  Select 
the  little  yellowish-colored  masses,  if  present,  remove  them  by 
means  of  the  fine  forceps  or  platinum  loop,  and  spread  them 
on  the  cover  glass  in  a  thin  layer.  If  the  sputum  is  homoge- 
neous, make  the  preparations  the  same  as  from  cultures,  using  a 
small  loopful  of  the  liquid.  If  the  sputum  is  viscid,  it  is  neces- 
sary to  use  the  forceps  to  spread  the  film  on  the  cover  glass. 
When  dry,  the  films  are  fixed  by  passing  the  preparations 
through  the  flame,  after  which  they  are  ready  to  be  stained. 

Instead  of  using  cover  glasses,  it  is  the  practice  in  some  labo- 
ratories to  spread  the  sputum  in  a  thin  film  over  the  central 
part  of  a  slide ;  dry,  fix,  and  stain  as  with  the  cover  glasses. 
The  water  is  dried  off  by  using  filter  or  blotting  paper,  and  the 
preparation  examined  without  a  cover  glass.  The  method  is 
said  to  be  easier  and  quicker  than  the  other,  and  the  cleaning 
of  cover  glasses  is  saved. 


BACTERIUM  TUBERCULOSIS  93 

135.  Gabbett*s  method  of  staining  tubercle  bacteria. 

1.  The  stain  (carbol  fuchsin)  : 

Fuchsin i  gram 

Absolute  alcohol lo  cc. 

5%  carbolic  acid loo  cc. 

2 .  The  decolorizer  and  counter  stain  : 

Methylene-blue  powder 2  grams 

io%  sulphuric  acid loo  cc. 

Stain  the  preparation  with  the  first  solution  as  described 
(§  100),  then  rinse  in  water  and  stain  i  minute  with  the  second 
solution,  which  decolorizes  and  counterstains  at  the  same  time, 
and  again  rinse  in  water.  If  the  film  has  a  bluish  tint,  it  is 
ready  for  examination ;  if  not,  it  should  be  stained  a  little  longer 
in  the  second  solution.  In  these  preparations  the  tubercle 
bacteria  should  appear  as  slender,  more  or  less  curved,  rod- 
shaped  bodies  of  a  deep  reddish  color,  while  the  surrounding 
tissue  and  other  bacteria  present  are  stained  a  more  or  less 
intense  blue. 

Sudan  III  is  reported  by  Dorset  to  be  a  very  good  differen- 
tial stain  for  this  organism.  A  saturated  solution  in  8o%  alco- 
hol is  used.  It  is  reported  to  be  effective  in  differentiating  the 
tubercle  organism  from  that  of  leprosy  and  from  the  smegma 
bacillus. 


94  LABORATORY  BACTERIOLOGY 

EXERCISE   XXXVII 

BACTERIUM   MALLEI 

136.  This  organism  grows  most  characteristically  on  potato 
and  somewhat  feebly  in  the  other  media  heretofore  used.  It 
develops  readily  on  acid  agar  and  in  acid  glycerin  agar  and 
acid  glycerin  bouillon.  For  this  reason  it  is  not  inoculated 
into  all  of  the  media.  In  diagnosing  glanders  it  is  customary 
to  inoculate  guinea  pigs  with  the  suspected  material  (see 
Appendix  III).  From  the  lesions  in  these  animals,  if  the  dis- 
ease develops,  pure  cultures  can  usually  be  obtained.  It  can 
be  identified  by  its  morphologic  and  cultural  characters. 

References.  Chapters  on  Bacteriu7ii  mallei  in  text-books. 
Smith,  The  Jour,  of  Comp.  Med.  and  Vet.  Archives,  March,  1890. 
De  Schweinitz  and  Dorset,  Jour,  of  the  Am.  Chem.  Soc,  Vol. 
XVII,  1898.  Finkelstein,  Centralb.  f.  Bakteriologie  u.  Parasi- 
tenkunde,  Bd.  XI,  1892,  S.  433.  Frothingham,  Proceedings  Am. 
Vet.  Med.  Asso.,  1901,  p.  360  (Straus'  method  of  diagnosing 
glanders).     See  recent  text-books. 

137.  Work  for  this  exercise.  Inoculate  a  tube  of  potato, 
one  of  agar,  one  of  acid  agar,  one  of  acid  glycerin  agar,  one 
of  glucose  agar,  one  of  bouillon,  and  one  of  acid  glycerin 
bouillon  from  a  culture  furnished.  (The  special  media  here 
introduced  will  be  furnished  by  the  instructor.) 

Stain  cover-glass  preparations  (furnished)  made  from  the 
lesions  in  guinea  pigs  which  were  inoculated  with  this  organ- 
ism. Stain  one  with  alkaline  methylene  blue  and  one  with  car- 
bol  fuchsin.  Note  especially  the  morphology  and  the  extent 
to  which  the  organisms  take  the  stain. 

Reexamine  the  cultures  of  avian  tubercle  and  complete  the 
notes  on  the  same. 


BACTERIUM   MALLEI  95 

EXERCISE  XXXVIII 

BACTERIUM   MALLEI  {continued) 

138.  Work  for  this  exercise.  Examine  and  carefully  describe 
all  the  cultures  of  Bacterium  mallei. 

Make  2  cover-glass  preparations  from  the  acid  agar  and 
from  the  bouillon  cultures,  and  stain  one  of  each  with  alkaline 
methylene  blue  and  one  with  carbol  fuchsin.  Describe  the 
bacteria  and  make  a  drawing  of  a  few  of  them.  Preserve  i 
preparation.  Keep  the  cultures  and  reexamine  them  at  each 
of  the  3  following  exercises.  Note  especially  the  character 
and  color  of  the  growth  on  the  potato  and  in  the  acid  glycerin 
bouillon. 

Examine  carefully  the  lesions  produced  by  the  inoculation 
of  Bacterium  mallei  in  a  male  guinea  pig  (Straus'  method 
of  diagnosing  glanders). 

Inoculate  by  Liborius'  method  (§  141)  2  tubes  of  liquid  agar, 
one  sugar  free,  the  other  containing  glucose,  2  fermentation 
tubes,  one  sugar  free,  the  other  containing  i  %  glucose  bouillon 
from  a  culture  of  an  anaerobic  bacillus,  the  bacillus  of  mahg- 
nant  oedema  or  of  symptomatic  anthrax  is  preferable  (fur- 
nished), and  place  the  inoculated  tubes  in  the  incubator. 


96  LABORATORY   BACTERIOLOGY 

EXERCISE  XXXIX 

CULTURES  OF  ANAEROBIC  BACTERIA 

139.  Certain  bacteria  will  not  grow  in  the  presence  of  oxy- 
gen (atmosphere),  and  consequently  they  must  be  cultivated 
in  a  medium  from  which  the  air  has  been  expelled,  or  in  the 
presence  of  some  natural  gas  such  as  hydrogen.  While  certain 
bacteria,  like  those  of  symptomatic  anthrax,  tetanus,  and  malig- 
nant oedema,  require  the  absence  of  oxygen,  others,  like  Bacil- 
lus subtilis^  will  not  multiply  without  it.  There  are,  however, 
a  large  number  of  bacteria  which  are  able  to  multiply  independ- 
ently of  the  presence  or  absence  of  this  element.  In  reference 
to  oxygen  recjuirements  bacteria  are  grouped  as  follows  : 

Obligative  aerobic  bacteria  require  oxygen. 

Obligative  anaerobic  bacteria  require  the  absence  of  oxygen. 

Facultative  aerobic  bacteria  grow  best  in  the  absence  of 
oxygen,  but  will  grow  in  the  presence  of  air. 

Facultative  anaerobic  bacteria  grow  best  in  the  presence  of 
oxygen,  but  will  grow  in  its  absence. 

There  are  several  methods  of  cultivating  anaerobic  bacteria, 
but  as  a  rule  they  are  difficult  and  cannot  be  easily  handled  in 
an  elementary  course.  Two  of  the  simpler  processes,  however, 
will  be  tried. 

References.  See  text-books  on  bacteriology.  Hunziker,  Jour, 
of  Applied  Microscopy,  Vol.  V,  No.  3.  Gould,  Annals  of  Surgery, 
October,  1903. 

140.  Work  for  this  exercise.  Examine  and  carefully  de- 
scribe the  appearance  of  the  anaerobic  cultures  made  in  Exer- 
cise XXXVIII. 

With  the  wire  loop  remove  one  of  the  colonies  from  the 
depth  of  the  agar  culture  and  examine  it  microscopically  (i) 
in  a  hanging-drop  preparation  and  (2 )  in  a  stained  cover-glass 


CULTURES   OF  ANAEROBIC   BACTERIA  97 

preparation.  Stain  with  carbol  fuchsin.  Examine  microscop- 
ically in  similar  preparations  the  bacteria  from  one  of  the 
fermentation  tubes ;  describe  their  appearance  in  each  prepa- 
ration and  make  a  drawing  of  a  few  of  them. 

Note  the  appearance  of  the  cultures  inoculated  for  the  study 
of  the  gas  production. 

Inoculate  (Liborius'  method)  2  tubes  of  agar,  one  sugar 
free,  the  other  containing  i  %  glucose,  and  2  fermentation 
tubes,  one  containing  sugar-free  bouillon,  the  other  i  %  glucose 
bouillon  from  a  culture  of  B,  tetani  furnished. 

Read  carefully  in  the  text-books  the  methods  for  cultivating 
anaerobic  bacteria. 

141.  Culture  by  Liborius*  method.  Liquefy  2  tubes  of  agar 
and  carefully  pour  them  together.  After  this,  boil  the  medium 
for  at  least  5  minutes  to  expel  the  air,  cool  it  down  to  a 
temperature  of  40°  C,  and  then  inoculate  it  from  the  cul- 
ture of  an  anaerobic  organism  furnished,  after  which  cool  the 
medium  rapidly  by  standing  it  in  cold  water  until  it  is  *set.  In 
inoculating  the  tube  insert  the  loop  nearly  to  the  bottom  and 
stir  very  gently.  In  making  the  inoculations  care  must  be 
taken  not  to  introduce  air  by  shaking  the  liquid  medium. 
Place  the  culture  in  the  incubator. 

142.  The  fermentation  tubes  for  cultivating  anaerobic  bac- 
teria. If  these  tubes  of  bouillon  have  been  properly  sterilized, 
the  closed  branch  is  practically  free  from  atmosphere.  The 
obligatory  anaerobe  will  grow  in  the  closed  branch  only,  while 
the  facultative  anaerobe  will  grow  in  both  the  open  and  closed 
parts.  If  the  organism  is  a  gas  producer,  the  gas  will  force  the 
cloudy  liquid  from  the  closed  bulb  into  the  open  one,  clouding 
the  otherwise  clear  liquid.  To  avoid  the  possibility  of  error 
in  interpreting  these  growths  it  is  well  to  inoculate  a  tube 
containing  sugar-free  bouillon,  in  which  case  the  liquid  in  the 
open  bulb  should  remain  clear,  as  gas  will  not  be  formed. 

These  tubes  are  of  equal  value  in  testing  obligatory  and 
facultative  anaerobic  organisms. 


gS  LABORATORY  BACTERIOLOGY 

EXERCISE  XL 

BACILLUS   TETANI 

143.  The  bacilli  of  tetanus  or  their  spores  occur  in  nature  as 
common  inhabitants  of  the  soil  —  at  least  they  are  found  in  the 

'soil  in  certain  localities.  They  are  believed  to  be  more  numer- 
ous in  certain  places  where  manure  has  been  thrown  in  abun- 
dance. Bacillus  tetani  is  anaerobic  and  consequently  must  be 
cultivated  according  to  methods  necessary  for  such  bacteria 
(§§  141,  142).  In  its  effect  upon  the  animal  body  it  remains 
at  the  point  of  inoculation,  the  disease  being  produced  by 
the  toxin  elaborated  by  the  bacilli. 

References.  See  text-books.  Kitasato,  Zeit.  f.  Hygiene, 
Bd.  X.,  S.  267.  Wesbrook,  Jour.  Path,  and  Bact.,  Vol.  Ill,  p.  70. 
Kanthack  (morphology),  Ibid.^  Vol.  IV,  p.  452.  Vaillard  and 
Rouget '(etiology),  Ann.  de  I'lnst.  Pasteur,  T.  VII,  p.  755. 

144.  Work  for  this  exercise.  Carefully  examine  the  2  cul- 
tures of  tetanus  bacilli  made  in  the  last  exercise  and  describe 
their  appearance. 

Make  2  cover-glass  preparations  from  the  liquid  culture 
and  stain  them  with  carbol  fuchsin.  Examine  them  micro- 
scopically and  describe  their  appearance.  Make  a  drawing  of 
a  few  bacilH  magnified  1000  diameters.  Keep  these  cultures 
until  the  next  exercise,  when  they  should  be  reexamined, 
sterilized,  and  rejected. 

Inoculate  a  tube  of  each  medium  in  Groups  A  and  B  with 
Bacterium  anthracis  from  a  culture  furnished  for  study  at  the 
next  exercise. 

145.  Method  of  isolating  tetanus  bacilli.  Tetanus  bacilli 
rarely  extend  beyond  the  place  of  inoculation  into  the  body 
of  the  infected  individual  (man  or  lower  animal).  In  the 
local  lesion  there  are  almost  always  other  bacteria,  so  that 


BACILLUS   TETANI  99 

cultures  made  directly  from  the  lesions  are  usually  impure. 
I  have  found  that  very  often  pure  cultures  may  be  obtained  by 
inoculating  a  guinea  pig  with  the  pus  or  exudate  from  the  local 
lesion  and  making  cultures  from  the  local  lesions  in  the  guinea 
pig,  the  juices  of  the  body  having  destroyed  the  saprophytic 
bacteria  which  were  present  in  the  first  material. 

Kitasato  has  recommended  a  procedure  which  is  reported 
to  be  fairly  successful.  It  is  to  inoculate  a  tube  of  agar  with 
tissue  from  the  local  lesion,  and  after  it  has  grown  for  from  24 
to  48  hours  at  a  temperature  of  37°  C.  heat  the  tube  to  80°  C, 
which  kills  all  the  other  bacteria,  but  does  not  destroy  the 
tetanus  spores.  From  this  culture  anaerobic  cultures  are 
prepared. 


lOO  LABORATORY  BACTERIOLOGY 

EXERCISE    XLI 

BACTERIUM   ANTIIRACIS 

146.  Anthrax  is  a  disease  affecting  cattle  and  man.  Bac- 
terium anthracis  is  interesting  because  of  its  spores,  which  are 
very  resistant  to  disinfectants.  Because  of  its  striking  mor- 
phology it  can  be  differentiated  microscopically  in  fresh  tissues. 
In  tissues  some  hours  after  death  there  is  a  putrefactive 
organism  that  resembles  it  morphologically.  Bad.  anthracis 
is  readily  diagnosed  in  cultures. 

147.  Work  for  this  exercise.  Examine  and  describe  each 
of  the  cultures  of  this  organism  made  during  the  last  exercise. 

Examine  microscopically  the  bouillon  and  agar  cultures  in 
both  hanging-drop  and  stained  cover-glass  preparations. 

Measure  a  few  of  the  bacteria  in  a  stained  preparation  and 
make  a  drawing  of  them  magnified  looo  diameters. 

Make  a  series  of  2  agar  plates  from  the  bouillon  culture. 

Examine  sections  of  animal  tissue  containing  anthrax  bac- 
teria. Make  and  examine  a  few  cover-glass  preparations  from 
the  liver  of  an  animal  (guinea  pig  or  rabbit)  which  has  just 
died  of  anthrax.     (This  will  be  furnished  by  the  instructor.) 

In  notes  state  whether  or  not  a  microscopic  examination  of 
tissues  is  sufficient  to  make  a  diagnosis  of  anthrax. 


BACTERIUM  ANTHRACIS  lOI 

EXERCISE    XLII 

BACTERIUM  ANTHRACIS  {continued) 

148.  Work  for  this  exercise.  Reexamine  all  of  the  cultures 
of  Bacterium  afithracis  and  describe  any  changes  in  their 
appearance  which  may  have  taken  place. 

Examine  the  agar  culture  for  spores  in  a  hanging-drop  prep- 
aration and  in  a  stained  cover-glass  preparation.  Describe 
the  appearance  of  the  bacteria  and  spores  in  a  preparation 
from  each. 

Study  and  describe  the  appearance  of  the  colonies  on  the 
agar  plates.  Make  an  outline  drawing  of  a  few  of  the  surface 
and  deep  colonies. 

Reject  all  cultures  except  the  agar  plates,  which  may  be 
kept  until  the  next  exercise  for  further  observation  before 
rejecting.  (These  cultures  should  be  put  in  charge  of  the 
instructor,  who  will  see  that  the  spores  are  destroyed  before 
the  tubes  are  cleaned.) 

Inoculate  a  tube  of  each  medium  in  Groups  A  and  B  and 
also  a  tube  of  Loeffler's  blood  serum  and  glycerin  agar  with 
Bacterium  diphtherice  from  a  culture  furnished  for  study  at  the 
next  exercise. 

References.  Chapters  on  anthrax  in  recent  editions  of  text- 
books. Chester,  Report  Delaware  College  Agric,  Exp.  Station, 
July,  1895.  Moore,  Report  N.  Y.  State  Com.  of  Agr.,  1897. 
Emmerich  and  Saida,  Centralb.  f.  Bakter.  u.  Parasitenkunde,  Bd. 
XXVII,  S.  ^^e,  Bongert,  Ibid.,  Bd.  XXXIV,  S.  497-  Preisz,  Ibid., 
Bd.  XXXV,  S.  280. 


102  LABORATORY  BACTERIOLOGY 

EXERCISE    XLIII 

BACTERIUM   DIPHTHERIiE 

149.  The  bacterium  of  diphtheria  is  often  called  the  Klebs- 
Loeffler  bacillus.  It  is  the  specific  cause  of  diphtheria  in  man  ; 
but  it  is  not,  so  far  as  known,  the  cause  of  diphtheria  in 
pigeons  and  poultry.  It  is  found  in  the  throats  of  people 
suffering  with  diphtheria,  and  often  in  the  throats  and  noses 
of  those  who  have  been  exposed  to  it.  These  are  designated 
as  ^'  germ  cases."  Ordinarily  it  is  not  found  elsewhere  in  the 
body,  although  it  is  occasionally  discovered  in  the  internal 
organs  and  blood.  It  usually  remains  in  the  throat  for  some 
days  after  its  lesions  have  disappeared.  Its  appearance  in  the 
throat  lesions  is  made  use  of  in  diagnosing  the  disease.  For 
this  reason  it  is  especially  important  that  its  morphology,  as 
well  as  its  cultural  characters,  should  be  carefully  determined. 
Although  this  organism  grows  on  nearly  all  of  the  media  com- 
monly used,  its  development  is  more  rapid  and  its  growth  more 
characteristic  on  Loeffler's  blood  serum.  The  bacterium  of 
diphtheria  seems  to  be  modified  in  its  morphology  in  growing 
on  different  media  more  than  any  of  the  other  pathogenic  bac- 
teria. Particular  attention  should  be  given  to  its  morphology 
and  staining  properties. 

References.  Chapters  on  diphtheria  in  recent  editions  of 
text-books.  Loeffler,  Mitth.  aus  d.  Kais.  Gesundheitsamte,  Bd.  II. 
Biggs,  Park,  and  Beebe,  Scientific  Bulletin  No.  i,  Health  Dept. 
City  of  New  York,  1895.  Wesbrook  (varieties).  Jour.  Boston 
Soc.  Med.  Sciences,  Vol.  IV,  p.  75.  Report  Am.  Pub.  Health 
Asso.,  1899,  p.  546.  Hill  (branching  forms).  Ibid.,  p.  554. 
Bergey  (pseudo-diphtheria).  Publications  of  the  University  of 
Pennsylvania,  new  series,  No.  4,  1 898.  Smith  (toxin).  Trans,  of 
the  Asso.  of  Am.  Phys.,  1896.  (Current  medical  literature  con- 
tains many  articles  on  this  subject.)  Hill,  Report  Boston  Board 
of  Health,  1901. 


BACTERIUM   DIPHTHERIA  IO3 

150.  Work  for  this  exercise.  Examine  and  describe  the 
cultures  made  in  Exercise  XLII. 

Examine  the  serum,  glycerin  agar,  and  bouillon  cultures 
microscopically  in  stained  cover-glass  preparations.  Stain  with 
alkahne  methylene  blue.  The  preparation  should  be  stained 
for  fully  5  minutes  and  decolorized  for  a  few  seconds  with 
alcohol.  Examine  the  bouillon  culture  in  a  hanging-drop 
preparation. 

Examine  carefully  a  fresh  culture  made  directly  from  a  diph- 
theritic throat,  including  stained  cover-glass  preparations.^ 
Stain  with  alkaline  methylene  blue  and  by  Neisser's  method. 
(It  is  not  always  possible  to  obtain  these  cultures  at  this  par- 
ticular time,  in  which  case  the  examination  will  be  postponed 
until  they  are  available.) 

151.  Neisser's  method  of  staining  diphtheria  bacteria.  Neisser 
has  recently  recommended  the  following  method  of  staining, 
in  which  2  solutions  are  employed,  viz. : 

(a)  One  gram  of  methylene  blue  (Griibler's)  is  dissolved  in 
20  cc.  of  96%  alcohol,  which  is  then  mixed  with  950  cc.  of 
distilled  water  and  50  cc.  of  glacial  acetic  acid. 

(J?)  Two  grams  of  vesuvin  are  dissolved  in  i  litre  of  boiling 
distilled  water  and  filtered. 

The  cover-glass  preparations  are  stained  in  (a)  for  from  i 
to  3  seconds,  washed  in  water,  and  then  stained  in  {b)  for  from 
3  to  5  seconds,  again  washed  in  water,  dried,  and  mounted. 
Stained  in  this  manner  the  bacilli  are  brown,  and  contain  2, 
or  rarely  3,  but  never  more,  blue  corpuscles.  The  corpuscles 
are  oval,  not  round,  in  shape,  and  their  diameter  appears 
greater  than  that  of  the  bacilli  in  which  they  are  situated. 

1  Clinically,  Bacterium  diphtherice  is  to  be  differentiated  from  the 
pseudo-diphtheriae  organism  and  from  a  bacillus  which  has  been  found  in 
decayed  teeth,  and  which  is  said  to  resemble  very  closely  in  its  mor- 
phology the  Klebs-Loeffler  bacillus.  It  is  also  to  be  distinguished  from 
the  Xerosis  bacillus  isolated  by  Neisser.  For  detailed  descriptions  of 
these  organisms,  see  text-books. 


104        LABORATORY  BACTERIOLOGY 

EXERCISE  XLIV 

BACTERIUM   DIPHTHERIA   {continued) 

152.  Work  for  this  exercise.  Examine  microscopically,  in 
stained  cover-glass  preparations,  the  bacteria  from  the  glycerin 
agar  and  Loeffler's  blood- serum  cultures.  Stain  with  alkaline 
methylene  blue  and  note  especially  the  way  the  bacteria  stain. 
Stain  a  few  preparations  after  Neisser's  method  (the  staining 
solutions  will  be  furnished)  and  compare  with  the  methylene- 
blue  stain. 

Note  with  special  care  the  morphology  of  the  bacteria  and 
make  a  drawing  of  a  few  of  them.  Compare  the  preparations 
with  those  made  from  the  same  cultures  in  previous  exercise 
and  note  any  differences  in  the  morphology  of  the  bacteria. 

Examine  very  carefully  a  guinea  pig  (furnished)  which  has 
died  from  the  effect  of  inoculation  with  diphtheria  organisms. 

Expectorate  into  a  watch  glass  which  has  been  wiped  with 
a  cloth  moistened  in  a  5%  solution  of  carbolic  acid.  From 
this  sputum  inoculate  a  tube  of  bouillon  and  one  of  slant  agar, 
and  make  a  series  of  2  agar  and  one  of  2  gelatin  plate  cul- 
tures. Use  a  small  loopful  of  sputum  for  each  tube  culture 
and  the  same  for  the  first  tube  in  the  plate  series. 


THE   BACTERIA  OF  THE   MOUTH  I05 

EXERCISE   XLV 

THE   BACTERIA   OF   THE   MOUTH 

153.  In  studying  cultures  froms  the  throats  of  diphtheritic 
individuals  one  encounters  many  variations  in  the  species  of 
bacteria  other  than  those  of  diphtheria  which  are  present. 
The  same  condition  holds  true  with  the  microscopic  exami- 
nation of  sputum  for  the  tubercle  bacteria.  The  fact  has  been 
determined  that  the  organism  of  lobar  pneumonia  is  often 
found  in  the  human  saliva,  and,  furthermore,  the  bacterium 
of  swine  plague  (^Bacterium  septiccefnice  hemorrhagicce)  is  often 
in  the  upper  air  passages  of  a  large  percentage  of  healthy 
swine,  and  a  like  organism  is  found  in  cattle,  cats,  and  dogs. 
In  order,  however,  to  isolate  them,  it  is  usually  necessary  to 
resort  to  rabbit  inoculation. 

Much  attention  has  been  given  to  the  study  of  the  bacteria 
of  the  mouth,  and  it  seems  desirable  that  a  few  examinations 
should  be  made  for  the  purpose  of  learning  something  definite 
concerning  the  variety  of  species  which  are  normal  inhabitants 
of,  and  which  seem  to  be  somewhat  localized  in,  the  oral  cav- 
ity, and  consequently  which  may  be  encountered  in  seeking 
for  pathogenic  forms.  In  addition  to  those  forms  which  seem 
to  be  more  or  less  localized  on  the  mucosa  of  the  mouth,  there 
is  usually  present  in  the  oral  cavity  a  large  and  changing  variety 
of  bacteria  which  have  been  introduced  with  the  food. 

References.  Vicentini,  Bacteria  of  the  Sputa,  London,  1897. 
Miller,  Die  Mikroorganismen  des  Mundhohle,  Leipsic,  1889. 
David,  Les  Microbes  de  la  Bouche,   Paris,   1890. 

154.  Work  for  this  exercise.  Examine  carefully  and  describe 
fully  the  cultures  made  from  sputum  at  the  last  exercise. 

Make  a  hanging-drop  preparation  from  one  of  each  of  the 
different  kinds  of  colonies  and  describe  the  appearance  of  the 


I06        LABORATORY  BACTERIOLOGY 

organism.  Note  the  name  of  the  genus  to  which  each  colony 
belongs,  together  with  the  approximate  number  of  colonies  of 
each. 

Make  one  or  more  cover-glass  preparations  from  the  mouth 
and  stain  with  alkaline  methylene  blue.  Note  carefully  the 
varieties  of  bacteria  they  contain. 

Inoculate  from  the  unnamed  cultures  furnished  such  media 
as  the  requirements  of  the  next  exercise  demand. 

155.  Making  cover-glass  preparations  from  the  mouth.  These 
can  be  made  from  the  sputum,  expectorated  in  a  watch  glass, 
or  from  the  scrapings  from  the  tongue,  gums,  pharynx,  or  from 
the  base  of  the  teeth.  If  any  of  the  latter  sources  is  chosen, 
the  part  from  which  the  material  is  to  be  taken  should  be 
scraped  carefully  with  a  sterile  (flamed)  platinum  loop  or  with 
the  blunt  point  of  a  scalpel  or  other  stiff  instrument.  The 
scrapings  are  spread  on  cover  glasses  the  same  as  the  sputum. 


IDENTIFYING  BACTERIA  FROM   CULTURES       10/ 

EXERCISE   XLVI 

IDENTIFYING  BACTERIA   FROM   CULTURES 

156.  The  two  cultures  of  bacteria  assigned  for  identification 
belong  to  species  already  studied,  and  the  student  should 
identify  the  species  of  bacteria  in  them.  To  do  this  such 
media  should  be  inoculated  and  such  microscopic  examina- 
tions made  as  he  thinks  necessary.  The  notes  should  contain 
a  complete  record  of  the  work  and  the  reasons  for  the  identi- 
fications made.  This  exercise  affords  a  good  opportunity  to 
begin  the  use  of  manuals  and  text-books  in  identifying  species. 

157.  Work  for  this  exercise.  Identify  the  bacteria  in  the 
cultures  assigned  at  the  last  exercise.  Use  any  method  which 
seems  to  be  necessary.  In  the  laboratory  notes  give  reasons 
for  the  procedure  adopted. 

Reexamine  and  reject  the  cultures  of  Bad,  diphthericB, 


I08         LABORATORY  BACTERIOLOGY 


EXERCISE   XLVII 

ISOLATING  AND  IDENTIFYING  BACTERIA  FROM 
ANIMAL  TISSUES 

158.  In  making  a  bacteriologic  investigation  into  the  cause 
of  death  in  an  animal  or  man  it  is  necessary  to  make  cultures 
from  the  various  organs  and  the  blood  to  find  whether  or 
not  there  are  any  pathogenic  or  other  bacteria  present.  This 
necessitates  a  knowledge  of  making  cultures  from  animal  tis- 
sues. In  this  exercise  an  experimental  animal  (rabbit  or 
guinea  pig)  which  has  died  from  some  bacterial  disease  will 
be  provided.  The  purpose  of  this  examination  is  to  find  out 
what  that  disease  is.  To  save  animals,  each  student  will  make 
cultures  from  but  one  organ.  From  time  to  time  during  the 
course  opportunity  will  be  afforded  for  making  cultures  from 
variously  diseased  animal  tissues.^  Each  student  will  be  given 
opportunity  to  inoculate  one  or  more  animals  some  time  during 
the  course. 

159.  Work  for  this  exercise.  The  experimental  animal  fur- 
nished will  be  tied  out  on  a  post-mortem  tray  and  the  viscera 
exposed.  (Directions  for  the  post-mortem  examination  will 
be  given  in  the  course  in  pathology.) 

Inoculate  a  tube  of  bouillon,  one  of  agar,  and  a  fermenta- 
tion tube  of  glucose  bouillon  from  either  the  liver,  spleen,  or 
kidney.  (In  an  actual  investigation  of  an  unknown  disease, 
cultures  should  be  made  from  all  of  the  organs,  blood,  and 
lymphatic  glands.) 

Make  a  series  of  3  agar  plate  cultures  from  the  same  organ. 

Make  and  examine  2  cover-glass  preparations  from  the 
organ  from  which  the  cultures  were  made.  Stain  one  with 
alkaline  methylene  blue  and  one  with  carbol  fuchsin.     (It  is 

1  For  methods  of  inoculating  animals  for  purposes  of  diagnosis,  see 
Appendix  III. 


ISOLATING  BACTERIA  FROM  TISSUES  IO9 

sometimes  necessary  to  fix  pieces  of  the  tissue  in  alcohol  or 
in  some  other  fixing  fluid  for  sectioning  and  staining,  pre- 
paratory to  studying  them.) 

Preserve  one  of  the  cover-glass  preparations  to  accompany 
the  notes. 

160.  Making  cultures  from  animal  tissues.  Heat  a  platinum 
spatula  to  a  white  heat  in  a  gas  flame  and  scorch  the  surface 
of  the  organ.  Flame  a  pair  of  fine  forceps,  tear  an  opening 
through  the  scorched  surface,  and  crush  a  bit  of  tissue  under- 
neath it.  With  the  platinum  loop  take  up  a  loopful  of  the 
crushed  tissue,  with  which  inoculate  the  media.  It  is  also 
desirable  to  inoculate  a  tube  of  slant  agar  with  the  needle  by 
drawing  it  over  the  surface  of  the  medium  after  charging  it 
with  tissue.  In  making  plate  cultures  use  a  loopful  of  the 
crushed  tissue  for  the  first  tube.  The  quantity  of  the  tissue 
necessary  to  give  a  desired  number  of  colonies  cannot  be 
anticipated,  although  experience  in  working  with  different 
organisms  in  animals  renders  one  able  to  approximate  the 
amount  required. 


no  LABORATORY  BACTERIOLOGY 


EXERCISE  XLVIII 

ISOLATING  AND  IDENTIFYING   BACTERIA   FROM 
ANIMAL  TISSUES  {contifiued) 

161.  Work  for  this  exercise.  Examine  and  describe  all  of 
the  cultures  made  from  the  animal  tissues. 

Examine  the  bouillon  and  agar  cultures  microscopically  both 
in  the  fresh  condition  and  in  stained  cover-glass  preparations. 

If  the  species  cannot  be  determined  from  these  cultures 
and  examinations,  make  such  other  cultures  from  these  as 
may  be  necessary  to  enable  one  to  do  so. 

After  the  species  are  identified,  state  in  the  notes  the  facts 
upon  which  the  identification  is  made. 


EXAMINATION   OF  TISSUES  III 


EXERCISE  XLIX 

THE  EXAMINATION  OF  SECTIONS   OF  TISSUE 
CONTAINING   BACTERIA 

162.  The  preparation  of  tissues  for  sectioning  and  the  study 
of  the  tissue  changes  more  properly  belong  to  the  course  in 
pathology.  It  is  important,  however,  that  one  should  be  able 
to  distinguish  bacteria  in  the  lesions  which  they  produce. 
For  this  reason  an  exercise  is  devoted  to  the  study  of  bacteria 
in  sections  of  tissues  already  stained  and  mounted.  These 
include  the  various  pneumonias,  tuberculosis,  anthrax,  hog 
cholera,  typhoid,  septicaemia,  etc. 

163.  Work  for  this  exercise.  Examine  the  sections  fur- 
nished for  bacteria  and  note  especially  their  distribution  in 
the  tissues.  Make  drawings  of  a  few  of  the  bacteria  from 
each  preparation. 

Compare  the  bacteria  in  the  sections  with  the  cover-glass 
preparations  which  have  been  made  from  cultures  of  the  same 
species,  and  note  any  differences  in  their  appearance  which 
may  be  detected. 

Examine  all  cultures  for  identification  in  previous  exercises. 


112  LABORATORY  BACTERIOLOGY 


EXERCISE  L 

BACTERIOLOGIC  EXAMINATION  OF  PUS  AND 
EXUDATES 

164.  It  is  often  very  desirable  for  diagnostic  purposes  to 
make  a  bacteriologic  examination  of  the  pus  from  abscesses 
and  the  mucopurulent  discharges  or  exudates  from  mucous 
or  serous  membranes. 

Several  diseases  can  be  diagnosed  in  this  way.  It  is  often 
necessary  to  make  cultures  and  it  is  always  advisable  to  do  so 
whenever  the  material  is  in  a  suitable  condition.  Among  the 
specific  diseases  for  which  such  an  examination  is  especially 
valuable  are  actinomycosis,  gonorrhea,  diphtheria,  and  tuber- 
culosis. Further,  it  is  often  desirable  to  determine  the  genera 
of  the  bacteria  in  the  numerous  abscesses  and  suppurating 
wounds  encountered  in  both  man  and  the  lower  animals. 
Such  examinations  of  the  more  desirable  cases  will  be  made 
from  time  to  time  as  they  become  available.  In  this  exercise 
such  cover-glass  preparations  will  be  examined  as  have  been 
accumulated  for  this  purpose. 

165.  Work  for  this  exercise.  Examine  the  pus  in  the  fresh 
condition  and  note  its  composition,  leucocytes,  red  blood 
corpuscles,  fungi  (actinomycosis),  etc. 

Make  cover-glass  preparations  and  stain  one  or  more  of 
them  with  carbol  fuchsin  and  one  with  alkaline  methylene 
blue  and  examine.  Note  the  cellular  tissue  elements  present 
and  describe  the  bacteria  found.  If  the  pus  is  from  a  case 
suspected  to  be  of  a  specific  nature,  stain  and  examine  for  the 
corresponding  organism. 

If  actinomycosis,  the  ray  fungus  may  be  seen  better  in  the 
fresh  preparation.  Add  a  drop  of  a  io%  solution  of  caustic 
potash  to  a  loopful  of  pus  on  the  slide  and  cover  it  with  a 
cover  glass  and  examine. 


EXAMINATION   OF  PUS  AND  EXUDATES  II3 

If  gonorrheal  discharge,  stain  the  cover-glass  preparations 
with  an  alcoholic  solution  of  eosin  and  alkaline  methylene 
blue,  or  with  carbol  fuchsin.  Note  the  appearance  of  the 
cocci  both  within  and  outside  of  the  pus  cells. 

If  from  supposed  tuberculosis,  stain  for  that  organism. 

If  from  diphtheria,  stain  for  that  organism  and  note  the 
morphology  of  the  bacteria. 

If  from  the  pus  of  an  abscess,  stain  for  pyogenic  bacteria. 

166.  Making  cover-glass  preparations  from  pus.  Spread  as 
thin  a  film  of  the  pus  as  possible  on  the  cover  glass.  This  can 
be  readily  done  by  drawing  the  edge  of  a  square  cover  glass 
over  the  surface  of  another  cover  glass  on  which  a  bit  of  the 
pus  has  been  placed.  See  method  for  making  cover-glass 
preparations  from  blood  (§  122). 


114  LABORATORY   BACTERIOLOGY 


EXERCISE    LI 

A   BACTERIOLOGIC    EXAMINATION   OF   THE    SKIN   FOR 

MICROCOCCUS    EPIDERMIDIS    ALBUS    AND 

OTHER  BACTERIA 

167.  There  is  liable  to  be  on  or  in  the  skin  a  number  of  bac- 
teria which  resist  the  ordinary  methods  of  cleansing,  owing  to 
their  being  deeply  seated  in  the  epidermis.  The  most  impor- 
tant among  these  is  M,  {Staph.)  epidermidis  albus.  These 
organisms  often  infect  wounds  in  surgical  operations.  An 
abrasion  of  the  skin  with  a  sterile  instrument  may  be  followed 
by  the  infection  of  the  wound  with  this  or  other  species  of  bac- 
teria which  were  on  or  in  the  skin  itself.  The  work  of  this 
exercise  is  to  demonstrate  the  presence  of  these  organisms  on 
the  skin  of  supposedly  sterilized  hands. 

References.  Dennis'  System  of  Surgery,  Vol.  I,  p.  249. 
This  chapter,  written  by  Professor  Welch,  contains  a  summary  of 
the  present  knowledge  of  the  bacteria  of  the  skin,  with  references 
to  original  articles. 

168.  Work  for  this  exercise.  Wash  the  hands  thoroughly 
with  soap  and  water,  using  a  sterilized  brush.  Then  wash 
them  in  a  solution  of  i  to  1000  corrosive  sublimate  for  5 
minutes,  rinse  thoroughly  in  boiled  water,  and  wipe  with  a 
sterilized  towel  (furnished). 

With  a  flamed  and  cooled  scalpel  scrape  the  epidermis  over 
a  small  area  about  the  finger  nails,  and  with  these  scrapings 
inoculate  a  tube  of  bouillon  and  make  a  series  of  2  agar  plate 
cultures. 

Make  a  similar  series  of  cultures  with  the  scrapings  from 
the  back  or  palm  of  the  hand. 

At  the  next  exercise  describe  these  cultures  and  examine  the 
colonies  microscopically  to  determine  the  genera  of  bacteria. 


EXAMINATION   OF  THE   SKIN  IIS 

If  a  micrococcus  which  grows  in  clumps  is  found,  inoculate  a 
tube  of  agar  with  it,  and  at  the  following  exercise  examine  and 
describe  its  appearance.  Indicate  in  the  notes  the  number  of 
colonies  of  bacteria  which  developed  in  the  plate  cultures  and 
the  genera  which  appear  in  the  bouillon  culture. 


Il6  LABORATORY  BACTERIOLOGY 


EXERCISE    LII 

DETERMINING  THE  THERMAL  DEATH  POINT  OF 
BACTERIA 

169.  It  is  important  to  know  the  minimum  temperature 
which  will  kill  bacteria,  especially  the  pathogenic  forms.  The 
uses  to  which  such  knowledge  can  be  put  are  numerous  in 
practical  sanitary  medicine,  disinfection,  and  pasteurization. 
For  the  various  methods  employed  in  making  these  determi- 
nations, see  text-books  and  special  articles  on  this  subject. 
The  method  here  given,  and  which  can  be  followed  by  a  full 
section  of  students,  will  give  only  approximate  results.  It 
should  not  vary,  however,  more  than  one  degree  from  the 
actual  thermal  death  point  in  moist  heat  of  the  organisms 
tested.  In  this  exercise  students  may  work  in  groups  with 
satisfactory  results  and  with  the  saving  of  much  media. 

170.  Work  for  this  exercise.  Take  lo  tubes  of  bouillon, 
place  8  of  them  in  a  wire  basket,  and  stand  it  in  the  thermo- 
regulated  water  bath  at  6o°  C.  After  15  minutes  remove  the 
tubes  and  inoculate  4  of  them  from  a  culture  of  B,  subtilis 
and  4  of  them  from  a  culture  of  B.  typhosus  or  B,  cholercE  siiis 
(the  cultures  will  be  furnished).  In  inoculating  the  tubes  be 
sure  not  to  touch  the  sides  above  the  surface  of  the  bouillon 
with  the  wire. 

After  the  tubes  are  inoculated  return  them  to  the  water  bath 
adjusted  at  60°  C.  The  water  should  come  just  above  the 
liquid  in  the  tubes.  Remove  the  tubes,  one  of  each  species, 
as  follows:  one  in  5  minutes,  one  in  10  minutes,  one  in  15 
minutes,  and  one  in  20  minutes.  Label  and  place  them  in 
the  incubator. 

Inoculate  the  other  two  tulles  of  bouillon,  one  from  each  of 
the  cultures  used,  and  place  them  in  the  incubator  for  controls. 


THE   THERMAL   DEATH   POINT  OF   BACTERIA     11/ 

At  the  next  exercise  examine  the  heated  tubes  and  note 
which  are  clear  and  which  contain  a  growth.  If  the  tubes 
heated  for  lo  minutes  or  longer  have  a  growth,  repeat  the 
experiment  at  70°  C.  If  this  fails  to  destroy  them,  repeat  at 
80°  C,  and  if  necessary  apply  a  still  higher  temperature. 

Examine  the  cultures  microscopically  in  all  the  fertile  tubes 
to  determine  if  they  are  pure. 

Explain  the  cause  for  the  difference  in  the  thermal  death 
point  between  these  two  organisms. 

Examine  the  cultures  made  from  the  scrapings  of  the  skin 
in  the  previous  exercise. 


Il8        LABORATORY  BACTERIOLOGY 

EXERCISE    LIII 

DETERMINING  THE  EFFICIENCY  OF  DISINFECTANTS 

171.  The  efficiency  of  the  more  commonly  used  disinfect- 
ants has  been  determined  for  most  of  the  pathogenic  bacteria, 
but  new  disinfectants  are  constantly  being  put  upon  the  mar- 
ket, and  before  it  is  safe  to  use  or  recommend  them,  their  effi- 
ciency should  be  determined.  With  many  of  the  disinfectants, 
such  as  carbolic  acid,  corrosive  sublimate,  Hme,  and  the  min- 
eral acids,  much  stronger  solutions  are  commonly  used  than 
are  actually  necessary  to  kill  the  bacteria,  owing  to  the  fact 
that  frequently  it  is  necessary  to  allow  for  an  indefinite  waste 
due  to  the  union  of  the  disinfectant  with  other  substances, 
usually  organic,  with  which  the  bacteria  are  mixed.  For  the 
different  methods  of  testing  the  efficiency  of  disinfectants,  see 
text-books.     A  very  simple  process  is  given  here. 

It  may  be  desirable  for  students  to  work  in  groups  of  two  or 
more  in  order  to  economize  in  the  number  of  tubes  required. 
If  possible,  however,  each  student  should  make  all  of  the  tests. 

References.  Young,  Notes  on  Disinfectants  and  Disinfec- 
tion, Augusta,  1898.  Rideal,  Disinfection  and  Disinfectants, 
London.  Rosenau,  Disinfection  and  Disinfectants.  See  also 
text-books. 

172.  Work  for  this  exercise.  Put  10  cc.  of  a  2%  solution 
of  carbolic  acid,  prepared  from  sterile  distilled  water,  into  each 
of  2  sterile  test  tubes.  Add  to  one  of  these  tubes,  by  means  of 
a  sterilized  pipette,  .25  cc.  of  a  bouillon  culture  oi  B,  cholerce 
suis  or  B.  typhosus.  To  the  other  tube  add  a  like  quantity 
of  a  suspension  in  bouillon  or  sterile  water  of  an  agar  culture 
of  B,  subtilis  (furnished). 

Inoculate  a  tube  of  bouillon  containing  fully  7  cc.  with  6 
loopfuls  from  each  of  these  tubes  after  the  expiration  of  the 


THE  EFFICIENCY  OF  DISINFECTANTS  II 9 

following  periods  of  time  :  i  minute,  5  minutes,  10  minutes, 
and  30  minutes.  In  making  these  inoculations  allow  the  loop 
to  go  to  the  bottom  of  the  inoculated  tube.  Label  each  with 
the  strength  of  the  disinfectant  and  time  of  exposure  and 
place  it  in  the  incubator.  It  should  be  noted  that  the 
adding  of  .25  cc.  of  culture  diluted  slightly  the  strength  of 
the  disinfectant. 

Note  at  the  next  exercise  the  condition  of  each  inoculated 
tube.  From  them  the  approximate  strength  of  the  disinfectant 
used  and  the  time  necessary  to  destroy  the  bacteria  can  be 
determined.  When  this  is  found  the  more  exact  strength  and 
time  can  be  determined  by  repeating  the  experiment  with 
weaker  dilutions  or  shorter  exposures  or  both. 

Examine  cultures  that  were  heated  at  the  previous  exercise. 


120  LABORATORY   BACTERIOLOGY 

EXERCISE    LIV 

PASTEURIZING   AND   STERILIZING   MILK 

173.  Milk  is  pasteurized,  in  the  present  acceptance  of  the 
term,  when  all  of  the  pathogenic  bacteria  which  it  may  happen 
to  contain  (with  the  exception  of  the  spores  of  anthrax)  are 
destroyed,  with  the  more  important  saprophytes.  It  is  not 
necessarily  sterile,  although  it  sometimes  is.  The  temperature 
should  be  from  60°  to  68°  C.  and  the  time  for  heating  20  minutes. 

In  this  exercise  it  is  the  purpose  to  study  the  effect  of  this 
process  on  the  bacteria  of  milk  and  to  compare  its  effect  with 
that  of  sterilization. 

In  the  generally  accepted  use  of  the  term,  milk  is  sterilized 
when  it  has  been  boiled.  Milk,  however,  is  a  difficult  substance 
to  sterilize,  so  that  it  occasionally  happens  that  milk  which  has 
been  boiled  for  from  5  to  10  minutes  still  contains  living  organ- 
isms (spores). 

In  this  exercise  students  may  work  in  small  groups. 

174.  Work  for  this  exercise.  From  the  fresh  milk  provided, 
make  2  agar  plates,  using  i  and  2  loopfuls,  respectively,  of 
the  milk.  Put  25  cc.  in  each  of  6  large  test  tubes  and  set  one 
in  the  incubator  and  leave  one  at  the  room  temperature. 
Boil  two  of  them  for  30  minutes  in  a  closed  water  bath,  and 
pasteurize  the  remaining  two  by  heating  them  in  the  water  bath 
for  30  minutes  at  65°  C.  It  requires  about  10  minutes  for  the 
milk  in  the  tubes  to  reach  the  temperature  of  the  water,  leaving 
the  milk  exposed  to  the  temperature  of  the  water  for  20  min- 
utes. It  should  be  cooled  quickly  by  standing  the  tubes  in 
cold  water. 

After  the  tubes  are  cooled,  make  3  agar  plates  from  one  of 
the  tubes  treated  by  each  process,  using  i  loopful  of  milk  for 
the  first  plate,  3  loopfuls  for  the  second,  and  .25  cc.  (measure 
with  a  graduated  pipette)  for  the  third.    Place  in  the  incubator 


PASTEURIZING  AND   STERILIZING   MILK  121 

one  of  the  tubes  of  milk  treated  by  each  process  with  the 
plate  cultures,  and  leave  the  other  tubes  with  a  tube  of  the 
fresh  milk  at  the  room  temperature. 

At  the  next  exercise  note  carefully  the  condition  of  the  milk 
in  each  of  the  various  tubes  and  also  the  number  of  colonies 
on  the  agar  plates. 

Examine  the  cultures  made  in  Exercise  LIIL         * 


122  LABORATORY   BACTERIOLOGY 


EXERCISE    LV 

THE   QUANTITATIVE  BACTERIOLOGIC   EXAMINATION 
OF  WATER 

175.  This  is  to  determine  the  number  of  bacteria  in  water. 
In  preparing  media  for  this  purpose  the  directions  given  in  the 
Journal  of  the  American  PubHc'Heahh  Association  for  Janu- 
ary, 1898,  p.  60,  should  be  followed.  The  conditions  of  tem- 
perature and  of  media  which  favor  growth  differ  for  different 
species.  Many  bacteria  found  in  water  will  not  grow  at  the 
incubator  temperature,  while  others  which  may  be  in  it  grow 
very  slowly  at  the  room  temperature.  To  determine  numbers 
it  is  better  to  grow  the  bacteria  in  gelatin  plates  at  the  tem- 
perature of  the  room.  (In  an  actual  examination  a  much 
larger  number  of  plate  cultures  than  can  be  managed  here 
should  be  made.) 

In  this  exercise  students  may  work  in  small  groups. 

176.  Work  for  this  exercise.  Make  from  the  properly  col- 
lected water  4  gelatin  plates,  using  a  definite  quantity  of  water 
for  each.  To  begin  with,  it  may  be  safe  to  inoculate  these 
tubes  with  o.i,  0.25,  0.50,  and  i.oo  cc,  respectively. 

To  determine  if  there  are  gas-producing  bacteria,  and  the 
approximate  number  of  these  if  any,  inoculate  10  fermentation 
tubes  with  o.i  cc.  each  and  5  with  0.2  cc.  each  of  the  water. 
In  place  of  the  fermentation  tubes  glucose  agar  may  be  used. 
In  this  case  i  fermentation  tube  of  glucose  bouillon  should  be 
inoculated  with  3  cc.  of  the  water  to  determine  the  quantity 
of  gas  produced,  if  there  is  any.  If  a  large  fermentation  tube 
is  used,  add  5  cc.  of  the  water.  From  the  gas  produced  in 
these  tubes  determine  approximately  the  number  of  the  gas- 
producing  bacteria. 

Careful  and  full  notes  should  be  taken  on  this  examina- 
tion.    The  preliminary  methods  for  making  a  bacteriologic 


EXAMINATION  OF   WATER  1 23 

examination  have  already  been  given,  and  this  is  largely  in  the 
nature  of  an  investigation  by  each  student.  It  is  not  expected 
that  the  special  methods  other  than  those  used  in  the  labora- 
tory for  pathogenic  bacteria  will  be  tried. 

Examine  the  milk  tubes  of  the  preceding  exercise  and  the 
cultures  made  from  them. 

177.  Collecting  water.  If  the  water  is  collected  from  a 
spigot  or  pump,  allow  it  to  flow  for  2  or  3  minutes  first,  and 
then  collect  the  desired  quantity  (100-200  cc.)  in  a  sterile 
bottle  and  cork  tightly ;  if  near  at  hand,  absorbent  cotton 
plugs  may  be  used. 

If  from  a  stream  or  river,  withdraw  the  stopper  and  immerse 
the  sterile  bottle,  mouth  downward,  to  the  depth  desired  and 
allow  it  to  fill.  There  are  several  mechanical  devices  for  col- 
lecting water  from  considerable  depths  from  the  surface. 


124        LABORATORY  BACTERIOLOGY 

EXERCISE    LVI 

THE  QUALITATIVE  EXAMINATION  OF  WATER 

178.  The  qualitative  examination  of  water  consists  in  deter- 
mining the  species  of  bacteria  present.  From  a  sanitary  stand- 
point it  consists  in  finding,  if  present,  those  species  which  may 
be  the  cause  of  disease  among  people  or  animals  consuming  it. 
The  pathogenic  bacteria  which  may  be  in  the  water  will  depend 
upon  the  conditions  ;  but  usually  in  this  country  water  is  exam- 
ined for  typhoid  and  hog-cholera  bacilli  {B.  coli  communis  and 
Fs.  pyocyaneus). 

In  India  the  spirillum  of  Asiatic  cholera  may  be  found  in  the 
water.  Occasionally  anthrax  may  be  suspected.  It  should  be 
stated  that  Ps,fluorescens  liquefaciens,  pseudo- typhoid,  and  the 
transitional  forms  of  the  colon  group  are  to  be  carefully  dif- 
ferentiated from  Fs,  pyocyaneus  and  B,  typhosus.  Owing  to 
imperfect  descriptions  many  of  the  common  soil  and  water 
bacteria  cannot  be  readily  identified.  The  genera  are  all  that 
is  expected  here. 

References.  Frankland,  Micro-organisms  in  Water.  Fuller, 
Report  Am.  Public  Health  Asso.,  1899,  p.  580.  See  recent  edi- 
tions of  text-books  and  reports  of  the  Laboratory  Section  of  the 
Am.  Public  Health  Association  since  1900. 

179.  Work  for  this  exercise.  Examine  the  cultures  made 
in  Exercise  LV,  count  the  colonies  on  the  plates,  and  estimate 
from  them  the  number  of  bacteria  in  a  cubic  centimeter  of  the 
water ;  that  is,  if  there  are  40  colonies  on  the  plate  containing 
0.1  cc.  of  water,  there  are  400  bacteria  in  i  cc.  of  it. 

From  the  cultures  in  the  grape-sugar  media  estimate  the 
number  of  gas-producing  bacteria  present. 

Describe  the  appearance  of  the  different  colonies  and  indi- 
cate the  approximate  number  of  each  kind. 


EXAMINATION   OF   WATER  125 

Keep  the  plate  cultures  until  the  following "  exercise  and 
reexamine  and  count  the  colonies. 

Determine  the  obviously  different  genera  of  bacteria  by 
making  a  microscopic  examination  of  the  different  colonies. 

180.  Estimating  the  number  of  gas-producing  bacteria  in 
water.  If  there  is  gas  in  all  of  the  lo  fermentation  tubes 
inoculated  with  o.i  cc.  each,  it  would  show  that  there  were  lo 
or  more  of  these  bacteria  in  each  cubic  centimeter.  If  3  of 
the  5  tubes  inoculated  with  0.2  cc.  each  contained  gas,  it  would 
indicate  that  there  were  at  least  3  gas-producing  bacteria  in 
each  cubic  centimeter.  The  preliminary  results  must  be  veri- 
fied by  repeated  examinations. 


126  LABORATORY  BACTERIOLOGY 


EXERCISE    LVII 

EXAMINATION  OF  CERTAIN  BACTERIA  NOT   STUDIED 
IN   THE  LABORATORY 

181.  This  exercise  will  be  devoted  to  a  study  of  prepara- 
tions of  important  bacteria,  fungi,  and  pathogenic  protozoa  not 
cultivated  in  the  laboratory.  Unfortunately  the  number  neces- 
sarily omitted  is  large.  This  demonstration,  however,  will  aid 
in  fixing  in  the  mind  an  idea  of  the  morphology  of  these  forms 
which  may  be  of  some  assistance.  Certain  of  the  pathogenic 
fungi  and  protozoa,  such  as  the  ray  fungus  of  actinomycosis 
and  the  protozoa  of  Texas  fever  in  cattle  and  malaria  in  man 
will  also  be  demonstrated.  These  will  be  studied  more 
thoroughly  in  the  course  in  pathology. 

182.  Work  for  this  exercise.  Examine  and  make  draw- 
ings of  the  bacteria,  fungi,  and  protozoa  demonstrated  in  the 
preparations  furnished. 

Complete  and  hand  in  all  notes  on  laboratory  work. 


BACTERIOLOGICAL   DIAGNOSIS  1 2/ 

EXERCISE   LVIII 

BACTERIOLOGICAL  DIAGNOSIS 

183.  This  and  the  following  exercises  of  this  course  will  be 
devoted  to  the  application  of  the  methods  already  studied  to 
practical  diagnostic  work,  or  to  the  making  of  some  examina- 
tion or  examinations,  or  to  carrying  out  some  little  investiga- 
tion that  seems  best  suited  to  the  needs  of  the  student.  Just 
what  these  will  be  must  be  decided  by  the  instructor  at  the 
time.  We  have  found  very  helpful  the  diagnosis  of  tuberculosis, 
diphtheria,  anthrax,  pyogenic  bacteria,  and  others  from  mixed 
cultures  or  animal  tissues.  If  the  student  is  to  continue  work 
in  bacteriology,  a  study  of  the  varieties  of  some  group  of  bac- 
teria or  of  some  bacterial  flora  may  be  better.  It  is  important 
that  the  student  should  learn  something  of  the  possibilities  and 
limitations  of  the  methods  in  practical  bacteriological  work. 

184.  Work  for  this  and  the  following  exercises.  From  the 
material  furnished  make  such  examinations,  cultures,  or  other 
tests  as  in  your  opinion  are  called  for  to  make  the  determina- 
tions required. 

Make  careful  notes  on  the  work  and  state  fully  in  your 
report  the  reasons  for  the  conclusions  reached. 

At  or  before  the  last  exercise  have  all  apparatus  for  indi- 
vidual use  inspected  by  the  instructor  and  returned  to  the 
laboratory. 

185.  Staining  actinomyces.  This  fungus  is  stained  in  cover- 
glass  preparations  made  from  actinomycotic  tissue,  or  in  sec- 
tions by  any  of  the  basic  aniline  dyes.  Carbol  fuchsin  is  very 
good.  The  Gram  method  gives  very  excellent  results.  It  is, 
however,  not  easy  to  obtain  nicely  stained  preparations  show- 
ing both  the  ^'  clubs  "  and  the  mycelial  part  of  the  fungus. 

186.  Staining  blood  films  for  malarial  parasites.  Several 
methods    of    staining   blood    films    to    demonstrate    malarial 


128  LABORATORY   BACTERIOLOGY 

parasites  are  in  use,  but  the  following  (Nocht-Romanowsky) 
gives  the  most  uniform,  satisfactory  results. 

Preparation  of  stain,  i.  To  one  ounce  of  polychrome 
methylene  blue  (Griibler)  add  a  3%  solution  of  acetic  acid 
(U.S.P.,  33%)  drop  by  drop  until  it  no  longer  turns  red 
litmus  paper  blue  above  the  zone  coming  into  immediate 
contact  with  the  dye.     It  usually  requires  about  five  drops. 

2.  Make  a  saturated  (i  %)  watery  solution  of  methylene  blue, 
preferably  Ehrlich's  (Griibler)  or  Koch's,  dissolving  the  dye 
by  gentle  heat.  This  solution  improves  with  age,  and  should 
be  at  least  one  week  old. 

3.  Make  a  1%  watery  solution  of  Griibler's  watery  eosin. 
The  mixture  is  prepared  as  follows  : 

To  10  cc.  of  water  add  4  drops  of  the  eosin  solution,  6 
drops  of  neutralized  polychrome  blue,  and  2  drops  of  i  % 
methylene  blue,  mixing  well. 

Preparation  of  films  for  staining  malaria  organisms  in  the 
blood.  Clean  the  finger  or  ear  lobe  with  alcohol,  and  prick 
with  a  sterile  surgical  needle  or  lance  so  as  actually  to  incise 
the  capillaries.  The  blood  should  not  be  squeezed  out,  but 
should  flow  freely.  Wipe  away  the  first  two  or  three  drops. 
Apply  the  edge  of  one  end  of  a  glass  slide  to  a  small  drop 
of  blood  and  place  this  edge  on  the  surface  of  another  slide 
resting  on  a  firm  surface.  As  soon  as  the  drop  of  blood  has 
spread  along  the  line  of  contact,  holding  the  slide  at  an  angle 
of  about  30°,  draw  it  gently  along  the  surface  of  the  receiving 
slide,  spreading  the  blood  in  a  thin  film.  Allow  the  films  to 
dry  in  the  air  before  fixing. 

Fixing  the  blood  films,  i.  By  heat,  (^r)  Open  fiame.  Pass 
the  slide,  specimen  side  up,  slowly  through  the  flame  of  a 
Bunsen  burner  until  it  is  decidedly  too  hot  for  the  hand  to 
bear.  At  this  temperature,  which  probably  varies  from  110° 
to  150°  C.,  fixation  is  complete  in  from  i  to  2  minutes.  Over- 
heated slides  can  usually  be  seen  to  change  color  in  the  flame, 
after  which  the  red  cells  stain  yellowish  with  eosin.     A  httle 


BACTERIOLOGICAL  DIAGNOSIS  1 29 

practice  will  enable  one  to  tell  when  fixation  is  complete 
without  overheating,  {b)  Ovens,  Slides  may  be  placed  in 
an  oven  provided  with  a  thermometer  and  exposed  to  a  tem- 
perature of  from  110°  to  150°  C.  from  5  to  10  minutes. 

2.  By  alcohol.  Fix  in  from  95%  to  97%  alcohol  from  10 
to  30  minutes.  If  left  too  long  in  alcohol  they  do  not  stain 
so  well.  For  staining  malarial  parasites,  fixation  by  alcohol  is 
preferable. 

Method  of  usi?tg  the  stain.  Put  the  stain  in  a  Petri  dish, 
place  in  it  two  or  three  toothpicks,  matches  with  heads 
removed,  or  pieces  of  small  glass  rod  to  support  the  slides, 
and  place  them,  specimen  side  down,  in  the  stain  upon  this 
support.  This  allows  any  precipitate  to  settle  away  from 
rather  than  upon  the  slides.  Allow  the  stain  to  act  one  or 
two  hours.  They  will  not  overstain  in  twenty-four  hours. 
Wash  in  water  and  when  dry  they  are  ready  to  examine 
without  a  cover  glass.  Immersion  oil  can  be  applied  directly 
to  the  films  v/ithout  injury. 


APPENDIX 


REACTION   OF   CULTURE   MEDIA 

The  importance  of  the  reaction  of  media  as  a  controlling  factor 
in  the  development  of  biological  characters  is  of  so  much  importance 
that  the  methods  recommended  by  the  committee  of  bacteriologists 
appointed  in  1895  to  the  American  Public  Health  Association  in 
1897  are  appended  to  aid  those  who  may  not  have  the  transactions  of 
that  association  at  hand. 

^^The  first  thing  t  obtain  is  a  standard  indicator'  which  will 
give  uniform  results.  These  requirements  are  best  fulfilled  by  phe- 
nolphthalein. 

This  indicator  was  first  suggested  by  Schultze  in  combination  with 
the  titration  method  for  obtaining  the  desired  reaction  for  culture 
media  (Cent,  fur  Bakt.  und  Parasit.,  Bd.  X.,  1891,  S.  53),  but  its  gen- 
eral adoption  seems  to  have  been  retarded  largely  by  Dahmen  (Cent, 
fur  Bakt.  und  Parasit.,  Bd.  XII.,  1892,  S.  620),  who  claimed  that  its  use 
was  not  feasible,  owing  to  complications  which  might  arise  from  the 
presence  of  carbonates  and  ammonium  salts  in  the  solution  to  be 
tested.  These  objections  to  the  use  of  phenolphthalein  do  exist, 
but  may  be  readily  overcome. 

The  amount  of  free  and  combined  ammonia  present  in  culture 
media  at  the  time  the  reaction  is  determined,  has  been  found  not 
to  exceed  .003  %,  which  is  less  than  one-tenth  the  amount  which 
interferes  with  the  accuracy  of  this  indicator ;  while  the  production  ot 
carbon  dioxide  is  obviated  to  a  very  great  degree  by  neutralizing 
with  sodium  hydroxid  instead  of  with  sodium  carbonate,  and  any 
of  this  gas  which  may  be  absorbed  from  the  atmosphere  is  practi- 
cally all  driven  off  by  heat  during  the  preparation  of  the  media. 

The  great  advantage  in  the  use  of  phenolphthalein  over  other 
indicators  lies  in  the  fact  that  it  takes  into  account  the  reaction  of 

131 


132  LABORATORY  BACTERIOLOGY 

weak  organic  acids  and  of  organic  compounds  which  have  an  am- 
photeric reaction,  but  in  which  the  acid  character  predominates. 
Turmeric  possesses  the  same  properties,  but  the  change  in  color  from 
a  yellow  to  brown  is  less  satisfactory  than  the  development  of 
purple  red  color,  and  furthermore  turmeric  paper  changes  color 
rather  slowly,  while  with  phenolphthalein  the  color  appears  almost 
instantly. 

Another  advantage  to  be  gained  from  the  use  of  this  later  indi- 
cator is  its  behavior  toward  the  phosphates.  Petri  and  Maassen 
(Arbeiten  aus  dem  K.  Gesundheitsamte,  Bd.  VIIL,  1893,  S.  311) 
and  Timpe  (Cent,  fiir  Bakt.  und  Parasit.,  Bd.  XIV.,  1893,  S.  845  ; 
Bd.  XV.,  1894,  S.  394-664;  Bd.  XVII.,  1893,  S.  416)  have  shown 
that  the  amphoteric  reaction  of  media  is  associated  with  the  pres- 
ence of  phosphates,  and  that  there  are  present  in  peptone  and  gela- 
tin proteid  bodies  which  possess  both  an  acid  and  a  basic  nature, 
but  in  which  the  acid  character  predominates.  These  observers 
agree  that  to  determine  accurately  the  reaction  of  such  amphoteric 
compounds  phenolphthalein,  or  turmeric  paper,  should  be  used  as 
an  indicator. 

It  is  known  that  at  the  neutral  point  of  phenolphthalein  any  free 
phosphoric  acid  present  enters  into  combination,  and  the  mono- 
basic and  tribasic  salts  of  this  acid  are  changed  to  the  dibasic  form 
(NagHPG^).  Now  disodium  hydrogen  phosphate  reacts  alkaline 
to  litmus,  lacmoid,  rosolic  acid,  and  methyl-orange,  but  neutral  to 
phenolphthalein  and  turmeric. 

Studies  made  at  the  Lawrence  Experiment  Station  show  that  this 
acid  salt  may  be  added  to  culture  media  in  amounts  greatly  exceed- 
ing those  naturally  present  in  the  media  without  producing  any 
apparent  influence  upon  bacterial  development. 

From  these  facts  it  seems  clear  that  the  use  of  any  of  the  above- 
mentioned  indicators,  other  than  phenolphthalein  and  turmeric,  in 
the  presence  of  this  dibasic  phosphate,  prevents  the  addition  of  a 
sufficient  amount  of  free  alkali  to  effect  neutralization,  and  as  the 
amount  of  phosphates  in  media  varies  considerably,  the  reaction 
passes  beyond  accurate  control  when  litmus  and  other  substances  of 
its  class  are  used  as  indicators. 

Datum  point  to  which  all  degrees  of  reaction  shall  be  referred : 

From  the  available  evidence  it  seems  advisable  to  adopt  the  phe- 
nolphthalein neutral  point  as  the  fixed  point  to  which  all  degrees 
of  reaction  shall  be  referred. 


APPENDIX  133 

The  question  of  the  proper  reaction  of  media  for  the  cultivation 
of  bacteria,  and  the  method  of  obtaining  this  reaction,  have  been 
discussed  in  a  valuable  paper  by  Mr.  George  W.  Fuller,  published 
in  the  Journal  of  the  American  Public  Health  Association,  Vol.  20, 
Oct.,  1895,  p.  321.  Some  of  the  main  results  there  given  have  been 
mentioned  above. 

Method  of  determining  the  Degree  of  Reaction  of  Culture  Media.  — 
For  this  most  important  part  in  the  preparation  of  culture  media, 
burettes,  graduated  into  -^  c.c,  and  3  solutions  are  required. 

1.  A  .5  %  solution  of  commercial  phenolphthalein  in  50%  alcohol. 

2.  A  n/20  solution  of  sodium  hydroxid. 

3.  A  n/20  solution  of  hydric  chlorid. 

Solutions  Nos.  2  and  3  must  be  accurately  made  up  and  must  cor- 
respond with  the  normal  solutions  soon  to  be  referred  to.  Solutions 
of  sodium  hydroxid  are  prone  to  deterioration  from  the  absorption 
of  carbon  dioxid  and  the  consequent  formation  of  sodium  carbonate. 
To  prevent  as  much  as  possible  this  change,  it  is  well  to  place  in 
the  bottle  containing  the  stock  solution  a  small  amount  of  calcium 
hydroxid,  while  the  air  entering  the  burettes  or  the  supply  bottles 
should  be  made  to  pass  through  a  "  U  "  tube  containing  caustic  soda, 
to  extract  from  it  the  carbon  dioxid. 

The  medium  to  be  tested,  all  ingredients  being  dissolved,  is 
brought  to  the  prescribed  volume  by  the  addition  of  distilled  water 
to  replace  that  lost  by  boiling,  and  after  being  thoroughly  stirred, 
5  c.c.  are  transferred  to  a  6-inch  porcelain  evaporating  dish ;  to  this 
45  c.c.  of  distilled  water  are  added,  and  the  50  c.c.  of  fluid  are 
boiled  for  3  minutes  over  a  flame.  One  c.c.  of  the  solution  of 
phenolphthalein  (No.  i)  is  then  added,  and  by  titration  with  the 
required  reagent  (No.  2  or  3)  the  reaction  is  determined.  In  the 
majority  of  instances  the  reaction  will  be  found  to  be  so  that 
the  n/20  sodium  hydroxid  is  the  reagent  most  frequently  required. 
This  determination  should  be  made  not  less  than  three  times, 
and  the  average  of  the  results  obtained  taken  as  the  degree  of 
reaction. 

One  of  the  most  difficult  things  to  determine  in  this  process  is 
exactly  when  the  neutral  point  is  reached,  as  shown  by  the  color 
developed,  and  to  be  able  in  every  instance  to  obtain  the  same 
shade  of  color.  To  aid  in  this  regard,  it  may  here  be  remarked 
that  in  bright  daylight  the  first  change  that  can  be  seen  on  the 


134  LABORATORY  BACTERIOLOGY 

addition  of  alkali  is  a  very  faint  darkening  of  the  fluid,  which  on 
the  addition  of  more  alkali  becomes  a  more  evident  color,  and 
develops  into  what  may  be  described  as  an  Italian  pink.  A  still 
further  addition  of  alkali  suddenly  develops  a  clear  and  bright  pink 
color,  and  this  is  the  reaction  always  to  be  obtained. 

All  titrations  should  be  made  quickly  and  in  hot  solutions,  to 
avoid  complications  arising  from  the  presence  of  carbon  dioxid. 

When  this  manipulation  is  carried  out  uniformly,  as  here  sug- 
gested, and  the  end  point  having  the  same  intensity  of  color  is 
always  reached,  very  satisfactory  and  closely-agreeing  results  may 
be  obtained. 

Neutralization  of  Media.  —  The  next  step  in  the  process  is  to  add 
to  the  bulk  of  the  medium  the  calculated  amount  of  reagent,  either 
alkali  or  acid,  as  may  be  determined.  For  the  purpose  of  neutraliza- 
tion a  normal  solution  of  sodium  hydroxid  or  of  hydric  chlorid  is 
used,  and  after  being  thoroughly  stirred  the  fluid  thus  neutralized  is 
again  tested  in  the  same  manner  as  at  first  to  insure  the  proper 
reaction  of  the  medium  being  attained.  When  neutralization  is  to 
be  effected  by  the  addition  of  alkali,  it  not  infrequently  happens  that 
after  the  calculated  amount  of  normal  solution  of  sodium  hydroxid 
has  been  added  the  second  test  by  titration  will  show  that  the 
medium  is  still  acid  to  phenolphthalein,  to  the  extent  sometimes  of 
from  0.5  to  I  %.  This  discrepancy  is  perhaps  due  to  side  reactions, 
which  are  not  understood ;  the  reaction  of  the  medium,  however, 
must  be  brought  to  the  desired  point  by  the  further  addition  of 
sodium  hydroxid,  and  the  titrations  and  additions  of  alkali  must  be 
repeated  until  the  medium  has  the  desired  reaction  (i.e.  0.0  %  — 
0.005  %,  see  below). 

After  the  prescribed  period  of  heating  it  is  frequently  found  that 
the  medium  is  again  slightly  acid,  usually  about  0.5  %.  Without 
correcting  this  the  fluid  is  to  be  filtered  and  the  calculated  amount 
of  acid  or  alkali  is  to  be  added  to  change  the  reaction  to  the  one 
desired. 

A  still  further  change  in  reaction  is  not  infrequently  to  be  observed 
after  sterilization,  the  degree  of  acidity  varying  apparently  with  the 
composition  of  the  media  and  the  degree  and  continuance  of  the 
heat. 

Manner  of  expressing  the  Degree  of  Reaction  of  Culture  Media.  — 
Since  at   the   time   the  reaction  is  first  determined  culture  media 


APPENDIX  13s 

are  more  often  acid  than  alkaline,  it  is  proposed  that  acid  media  be 
designated  by  the  plus  sign  and  alkaline  media  by  the  minus  sign, 
and  that  the  degree  of  acidity  or  alkalinity  be  noted  in  parts  per 
hundred;  thus  a  medium  marked  +  1.5  would  indicate  that  the 
medium  was  acid  and  that  1.5  %  of  n/i  sodium  hydroxid  is  required 
to  make  it  neutral  to  phenolphthalein,  while  —1.5  would  indicate 
that  the  medium  was  alkaline  and  that  1.5  %  of  n/i  acid  must  be 
added  to  make  it  neutral  to  the  indicator. 

Limits  of  accuracy  of  the  proposed  method  for  the  control  of  the 
reaction'  of  media : 

The  available  data  are  as  yet  insufficient  to  warrant  any  conclu- 
sions upon  this  point.  The  limits  of  accuracy  seem  to  vary  with 
the  ingredients  employed  in  preparing  nutrient  media,  different 
samples  of  meat  infusion,  pepton,  and  gelatin  appearing  to  react 
differently  with  the  acids  and  alkalis  and  in  a  way  which  is  not 
understood. 

This  method,  nevertheless,  when  carefully  carried  out,  and  when 
the  media  before  titration  are  thoroughly  mixed  and  are  of  the 
prescribed  volume,  give  fairly  uniform  results. 

Standard  reaction  of  media  (provisional)  : 

Experience  seems  to  vary  somewhat  as  to  the  optimum  degree 
of  reaction  which  shall  be  uniformly  adopted  in  the  preparation  of 
standard  culture  media.  To  what  extent  this  is  due  to  variation  in 
natural  conditions  as  compared  with  variations  of  laboratory  pro- 
cedure, it  seems  impossible  to  state.  Somewhat  different  degrees 
of  reaction  for  optimum  growth  are  required,  not  only  in  or  upon 
the  media  of  different  composition  and  by  bacteria  of  different 
species,  but  also  by  bacteria  of  the  same  species  when  in  different 
stages  of  vitality. 

The  bulk  of  available  evidence  from  both  Europe  and  America 
points  to  a  reaction  of  +1.5  as  the  optimum  degree  of  reaction  for 
bacterial  development  in  inoculated  culture  media ;  and  while  this 
experience  is  at  variance  with  that  in  several  of  our  own  laboratories, 
it  has  been  deemed  wise  to  adopt  +1.5  as  the  provisional  standard 
reaction  of  media,  but  with  the  recommendation  that  the  optimum 
growth  reaction  be  always  recorded  in  species  descriptions." 

Journal  Am.  Public  Health  Association,  January,  1898. 


136  LABORATORY   BACTERIOLOGY 

II 

THE  OCULAR  MICROMETER  AND  MICROMETRY  1 

"  Ocular  Micrometer,  Eye-piece  Micrometer.  —  This,  as  the  name 
implies,  is  a  micrometer  to  be  used  with  the  ocular.  It  is  a  microm- 
eter on  glass,  and  the  lines  are  sufficiently  coarse  to  be  clearly  seen 
by  the  ocular.  The  lines  should  be  equidistant  and  about  j\^  or 
2V  mm.  apart,  and  every  fifth  line  should  be  longer  and  heavier  to 
facilitate  counting.  If  the  micrometer  is  ruled  in  squares  (net 
micrometer)  it  will  be  very  convenient  for  many  purposes. 

The  ocular  micrometer  is  placed  in  the  ocular,  no  matter  what  the 
form  of  the  ocular  {i.e.  whether  positive  or  negative),  at  the  level 
at  which  the  real  image  is  formed  by  the  objective,  and  the  image 
appears  to  be  immediately  upon  or  under  the  ocular  micrometer,  and 
hence  the  number  of  spaces  on  the  ocular 
micrometer  required  to  measure  the  real 
image  may  be  read  oif  directly.  This  is 
measuring  the  size  of  the  real  image,  how- 
ever, and  the  actual  size  of  the  object  can 
only  be  determined  by  determining  the  ratio 
between  the  size  of  the  real  image  and  the  Field  oi  large  filar  mi- 
object.      In  other    words,   it  is  necessary  to       crometer  showing  cross 

■'  '  ^  hairs     and     recording 

get  the  valuation  of  the  ocular  micrometer       comb. 

in  terms  of  a  stage  micrometer. 

Valuation  of  the  Ocular  Micrometer^  —  This  is  the  value  of  the 
divisions  of  the  ocular  micrometer  for  the  purpose  of  micrometry, 
and  is  entirely  relative,  depending  upon  the  magnification  of  the  real 
image  formed  by  the  objective  ;  consequently  it  changes  with  every 
change  in  the  magnification  of  the  real  image,  and  must  be  spe- 
cially determined  for  every  optical  combination  (i.e.  objective  and 
ocular)  and  for  every  change  in  the  length  of  the  tube  of  the  mi- 
croscope, that  is,  it  is  necessary  to  determine  the  ocular  microm- 
eter valuation  for  every  condition  modifying  the  real  image  of  the 
microscope  (152). 

1  These  paragraphs  are  from  Professor  S.  H.  Gage's  work  on  the  microscope, 
published  here  by  his  consent.  The  references  to  sections  are  to  the  seventh 
edition  of  The  Microscope, 


L*X  .X.XV,^    V^.*»V.W^.,      ... 


APPENDIX 


^2,7 


Any  Huygenian  ocular  may,  however,  be  used  as  a  micrometer 
ocular  by  placing  the  ocular  micrometer  at  the  level  of  the  ocular 
diaphragm,  where  the  real  image  is  formed.  If  there  is  a  slit  in  the 
side  of  the  ocular,  and  the  ocular  micrometer  is  mounted  in  some 
way,  it  may  be  introduced  through  the  opening  in  the  side.  When 
no  side  opening  exists,  the  mounting  of  the  eye-lens  may  be  un- 
screwed and  tlie  ocular  micrometer,  if  on  a  cover-glass,  can  be  laid 
on  the  upper  side  of  the  ocular  diaphragm. 

Obtaining  the  Valuation  of  the  Filar  Micrometer.  —  This  microm- 
eter (Figs.  98-99)  consists  of  a  Ramsden's  ocular  and  cross  lines. 


Filar  Micrometer. 


As  seen  in  Fig.  98  there  are  three  lines.  The  horizontal  and  one 
vertical  line  are  fixed.  One  vertical  line  may  be  moved  by  the 
screw  back  and  forth  across  the  field. 

For  obtaining  the  valuation  of  this  ocular  micrometer  an  accurate 
stage  micrometer  must  be  used.  Carefully  focus  the  ^  J-^  mm.  spaces. 
The  lines  of  the  ocular  micrometer  should  also  be  sharp.  Jf  they 
are  not,  focus  them  by  moving  the  top  of  the  ocular  up  or  down 
(164).  Make  the  vertical  lines  of  the  filar  micrometer  parallel 
with  the  lines  of  the  stage  micrometer.  Take  the  precautions 
regarding  the  width  of  the  stage  micrometer  lines  given  in  167. 


138  LABORATORY   BACTERIOLOGY 

Note  the  position  of  the  graduated  wheel  and  of  the  teeth  of  the 
recording  comb,  and  then  rotate  the  wheel  until  the  movable  lines 
traverse  one  space  on  the  stage  micrometer.  Each  tooth  of  the 
recording  comb  indicates  a  total  revolution  of  the  wheel,  and  by 
noting  the  number  of  teeth  required  and  the  graduations  on  the 
wheel,  the  revolutions  and  parts  of  revolution  required  to  measure 
the  3-J-jj  mm.  of  the  stage  micrometer  can  be  easily  noted.  Measure 
in  like  manner  four  or  five  spaces  and  get  the  average.  Suppose 
this  average  is  ij  revolutions,  or  125  graduations,  on  the  wheel,  to 
measure  the  yj^  mm.,  or  lo/x  (157),  then  one  of  the  graduations  on 
the  wheel  would  measure  lo/x  divided  by  i25=:.o8/x.  In  using  this 
valuation  for  actual  measurement,  the  tube  of  the  microscope  and 
the  objective  must  be  exactly  as  when  obtaining  the  valuation  (165). 

Example  of  Measurement.  —  Supposing  one  used  the  red  blood 
corpuscles  of  a  dog,  or  monkey,  etc.,  every  condition  being  as  when 
the  valuation  was  determined,  one  notes  very  accurately  how  many 
of  the  graduations  on  the  wheel  are  required  to  make  the  movable 
lines  traverse  the  object  from  edge  to  edge.  Suppose  it  requires  94 
of  the  graduations  to  measure  the  diameter,  the  actual  size  of  the 
corpuscle  would  be  94  x  .o8/a  =  7.52/x. 

The  advantage  of  the  filar  micrometer  is  that  the  valuation  of  one 
graduation  being  so  small,  even  the  smallest  object  to  be -measured 
would  require  several  graduations  to  measure  it.  In  ocular  microm- 
eters with  fixed  lines  small  objects  like  bacteria  might  not  fill  even 
one  space  ;  therefore  estimations,  not  measurements,  must  be  made. 
For  large  objects,  like  most  of  the  tissue  elements,  the  ocular  mi- 
crometers with  fixed  lines  answer  very  well,  for  the  part  which  must 
be  estimated  is  relatively  small,  and  the  chance  of  error  is  corre- 
spondingly small. 

Obtaining  the  Ocular  Micrometer  Valuation  for  an  Ocular  Microm-i 
eter  with  Fixed  Lines  (Figs.  33,  34,  p.  25).  —  Use  the  stage  microm- 
eter as  object.  Light  the  field  well  and  look  into  the  microscope. 
The  lines  of  the  ocular  micrometer  should  be  very  sharply  defined. 
If  they  are  not,  raise  or  lower  the  eye-lens  to  make  them  so,  that  is, 
focus  as  with  the  simple  magnifier. 

When  the  lines  of  the  ocular  micrometer  are  distinct,  focus  the 
microscope  (45,  46,  56)  for  the  stage  micrometer.  The  image  of 
the  stage  micrometer  will  appear  to  be  directly  under  or  upon  the 
ocular  micrometer. 


APPENDIX  139 

Make  the  lines  of  the  two  micrometers  parallel  by  rotating  the 
ocular  or  changing  the  position  of  the  stage  micrometer,  or  both  if 
necessary,  and  then  make  any  two  lines  of  the  stage  micrometer 
coincide  with  any  two  on  the  ocular  micrometer.  To  do  this  it  may 
be  necessary  to  pull  out  the  draw  tube  a  greater  or  less  distance. 
See  how  many  spaces  are  included  on  each  of  the  micrometers. 

Divide  the  value  of  the  included  space  or  spaces  on  the  stage 
micrometer  by  the  number  of  divisions  on  the  ocular  micrometer 
required  to  include  them,  and  the  quotient  so  obtained  will  give  the 
valuation  of  the  ocular  micrometer  in  fractions  of  the  unit  of  meas- 
ure of  the  stage  micrometer.  For  example,  suppose  the  millimetre 
is  taken  as  the  unit  for  the  stage  micrometer,  and  this  unit  is  divided 
into  spaces  of  xV  ^^^  rio  ^'^^'  I^  i^ow,  with  a  given  optical 
combination  and  tube  length,  it  requires  ip  spaces  on  the  ocular 
micrometer  to  include  the  real  image  of  xV  nim.  on  the  stage 
micrometer,  obviously  i  space  on  the  ocular  micrometer  would 
include  only  one-tenth  as  much,  or  -^^  mm.  lo^yj^  mm.,  that  is, 
each  space  on  the  ocular  micrometer  would  include  j^^  of  a  milli- 
metre on  the  stage  micrometer,  or  yj-  mm.  of  length  of  any 
object  under  the  microscope,  the  conditions  remaining  the  same. 
Or,  in  other  words,  it  would  require  100  spaces  on  the  ocular 
micrometer  to  include  i  mm.  on  the,  stage  micrometer,  then  as 
before  i  space  of  the  ocular  micrometer  would  have  a  valuation  of 
j^jj  mm.  for  the  purposes  of  micrometry;  and  the  size  of  any 
minute  object  may  be  determined  by  multiplying  this  valuation  of 
I  space  by  the  number  of  spaces  required  to  include  it.  For 
example,  suppose  the  fly's  wing  or  some  part  of  it  covers  8  spaces 
on  the  ocular  micrometer,  it  would  be  known  that  the  real  size  of 
the  part  measured  is  j-J-jj^  mm.  x  S^yfo  mm.  or  80/4  (157). 

Varying  the  Ocular  Micrometer  Valuation. — Any  change  in  the 
objective,  the  ocular,  or  the  tube  length  of  the  microscope,  that  is  to 
say,  any  change  in  the  size  of  the  real  image,  produces  a  corre- 
sponding change  in  the  ocular  micrometer  valuation  (152,  161)." 


I40  LABORATORY   BACTERIOLOGY 

III 

ANIMAL  INOCULATION   FOR   PURPOSES   OF   DIAGNOSIS 

It  is  not  always  possible  by  the  ordinary  culture  methods  to  suc- 
cessfully determine  the  specific  nature  of  a  disease  from  a  small 
piece  of  affected  organ  or  tissue  of  the  diseased  animal  or  man.  In 
making  a  positive  diagnosis,  therefore,  it  is  often  necessary  to  resort 
to  animal  inoculation.  This  is  done  by  injecting  into  the  animal 
chosen  a  small  quantity  of  the  tissue  or  fluid  supposed  to  contain 
the  virus  of  the  specific  disease,  such  as  tuberculosis,  glanders, 
rabies,  and  often  of  swine  plague,  hog  cholera,  anthrax,  diphtheria, 
and  others.  Animal  inoculation  is  further  demandatory  in  determin- 
ing the  degree  of  virulence  of  pathogenic  bacteria,  the  strength  of 
toxins,  antitoxins,  etc.  In  other  words,  the  living  animal  must  for 
the  present  serve  in  certain  instances  as  a  testing  reagent.  The  fact 
should  be  kept  in  mind  that  the  lesions  produced  in  the  experimental 
animal  are  not  necessarily  and  in  most  cases  they  are  not  the  same 
as  those  in  the  animal  (or  man)  from  which  the  virus  was  obtained. 
It  is  the  rule,  however,  that  each  virus  produces  characteristic 
lesions  from  which  the  disease  can  usually  be  diagnosed  in  the 
smaller  animal. 

Animals  Used.  —  For  simple  diagnostic  work  the  guinea  pig  and 
rabbit  are  usually  employed,  although  white  and  gray  mice,  dogs,  and 
other  animals  are  sometimes  used. 

Method.  —  In  preparing  the  animal  for  inoculation  the  hair  should 
be  removed  over  the  area  of  operation  by  the  use  of  scissors,  and  the 
skin  washed  and  disinfected.  A  solution  of  corrosive  sublimate, 
I  to  I  GOO,  or  a  5%  solution  of  carbolic  acid,  may  be  used.  The 
incision  should  be  made  with  a  sharp  knife.  Liquid  material  is 
usually  injected  with  a  hypodermic  syringe.  An  anaesthetic  should 
be  given  whenever  the  pain  inflicted  is  to  be  long  continued  or 
excessively  severe.  The  place  of  inoculation  should  be  chosen 
where  a  local  swelling,  infiltration  of  tissue,  or  abscess  would  not 
interfere  with  the  animal's  locomotion. 


APPENDIX  141 

SPECIFIC   DISEASES  FOR  WHICH   ANIMAL    INOCULATIONS  ARE    MOST 
COMMONLY  RESORTED   TO   FOR   DIAGNOSTIC   PURPOSES 

Tuberculosis.  —  Guinea  pigs  are  preferable,  although  rabbits  may 
be  used.  With  tuberculous  tissues  either  of  two  methods  may  be 
employed,  (i)  A  small  piece  (about  the  size  of  a  pea  or  bean)  of 
the  tissue  may  be  inserted  under  the  skin  by  first  making  an  incision 
with  a  sharp  scalpel  through  the  skin  and  superficial  fascia  and  then 
with  a  pair  of  fine  forceps  insert  the  bit  of  tissue  well  under  the  skin 
and  close  the  opening  with  one  or  more  sutures.  (2)  The  tissue 
may  be  crushed  in  a  mortar  and  thoroughly  mixed  with  a  few  cubic 
centimetres  of  sterile  water  or  bouillon  and  then  injected  with  a 
hypodermic  syringe.  The  needle  should  be  of  large  calibre.  If  it 
is  suspected  milk,  it  may  be  injected  into  the  abdominal  cavity.  If 
the  material  is  tuberculous  and  contains  living  tubercle  bacteria,  the 
death  of  the  animal  follows  in  from  three  weeks  to  four  months. 
Usually  the  lymphatic  glands  of  the  groin  and  axilla  are  enlarged 
and  often  caseous.  If  a  guinea  pig  is  used,  the  liver,  spleen,  lungs, 
and  kidneys  are  liable,  in  the  order  named,  to  be  affected  ;  if  a  rabbit, 
the  lungs  are  often  the  first  of  the  visceral  organs  to  be  attacked. 
(See  pathology  for  description  of  tissue  changes.) 

Glanders.  —  Male  guinea  pigs  should  be  used.  The  material  usu- 
ally consists  of  the  nasal  discharge  from  the  suspected  glandered 
horse,  or  bits  of  scrapings  from  the  ulcers,  or  pieces  of  affected 
tissue.  The  method  to  be  followed  is  precisely  the  same  as  with 
the  subcutaneous  injection  of  tuberculous  material.  In  these  cases 
there  is  liable  to  be  a  local  swelling  and  abscess.  The  first  indica- 
tion of  glanders  noticed  is  usually  orchitis.  The  lymphatic  glands 
in  the  groin  are  also  enlarged.  After  the  orchitis  becomes  well 
marked  the  guinea  pig  may  be  chloroformed  and  examined.  Pure 
cultures  of  the  specific  organism  can  be  obtained  in  most  cases  from 
the  suppurating  focus  in  the  testicle.  The  spleen  is  usually  en- 
larged and  sprinkled  with  grayish  nodules.  Other  organs  may  be 
involved. 

Rabies.  — The  method  usually  followed  in  diagnosing  rabies  is  to 
inoculate  a  rabbit,  guinea  pig,  or  dog  beneath  the  dura  with  a  bit 
of  the  brain  or  spinal  cord  of  the  suspected  rabid  animal.  Other 
methods  are  being  introduced  and  the  guinea  pig  is  reported  by 
some  to  respond  more  promptly,  but  in  my  experience  the  subdural 


T42  LABORATORY   BACTERIOLOGY 

inoculation  of  rabbits  has  been  most  reliable.  The  injection  through 
the  optic  foramen  has  been  tried.  The  subdural  method  is,  briefly 
?  stated,  as  follows  : 
I  The  brain  of  the  suspected  animal  is  removed  with  aseptic  pre- 
1  cautions  as  soon  as  possible  after  death.  A  small  piece  of  the  brain 
or  spinal  cord  is  placed  in  a  sterile  mortar  and  thoroughly  ground 
with  a  few  cubic  centimetres  of  sterilized  water  or  bouillon.  This 
forms  the  suspension  to  be  injected.  The  hands  of  the  operator  and 
all  instruments  are  carefully  disinfected.  The  rabbit  is  etherized, 
the  hair  clipped  from  the  head  between  the  eyes  and  ears,  and  the  skin 
thoroughly  washed  and  disinfected.  A  longitudinal  incision  is  then 
made,  the  skin  and  subcutaneous  tissue  held  back  by  means  of  a 
tenaculum,  a  crucial  incision  is  made  in  the  periosteum  on  one  side  of 
the  median  line  to  avoid  hemorrhage  from  the  longitudinal  sinus, 
and  the  four  corners  of  the  periosteum  reflected  or  pushed  back. 
By  the  aid  of  a  trephine  a  small  button  of  bone  is  easily  removed, 
leaving  the  dura  mater  exposed.  With  a  hypodermic  syringe  a 
drop  or  more  of  the  rabid  brain  suspension  is  injected  beneath  the 
dura,  the  periosteum  is  replaced,  the  skin  carefully  sutured  and  dis- 
infected, and  the  rabbit  returned  to  its  cage.  As  soon  as  the  influ- 
ence of  the  anaesthetic  has  passed  oif,  the  rabbit  shows  no  appearance 
of  discomfort.  If  the  operation  is  performed  in  the  forenoon,  the 
animal  partakes  of  its  evening  meal  with  the  usual  relish.  The 
inoculation  wound  heals  rapidly  and  the  rabbit  exhibits  every  ap- 
pearance of  being  in  perfect  health  until  the  beginning  of  the  specific 
symptoms,  which  occur  ordinarily  in  from  15  to  30  days,  usually 
in  about  20  days  after  the  inoculation.  Occasionally  the  symptoms 
appear  earlier  than  15  days  and  in  some  cases  the  rabbits  are  not 
attacked  for  from  i  to  3  months. 

The  symptoms  following  the  inoculations  have  in  my  experience 
been  quite  uniform,  the  only  pronounced  difference  being  in  the 
length  of  time  the  rabbits  lived  after  the  initial  manifestation  of 
the  disease.  The  fact  should  be  clearly  stated  that  rabbits  do 
not  ordinarily  become  furious.  In  some  instances  they  are  some- 
what nervous  for  a  day  or  two  preceding  the  paralysis.  There 
appears  to  be  a  marked  hyperaesthesia.  Usually  the  first  indica- 
tion of  the  disease  is  a  partial  paralysis  of  one  or  both  hind  limbs. 
This  gradually  advances  until  the  rabbits  are  completely  pros- 
trated, the  only  evidence  of  life  being  a  slight  respiratory  move- 


APPENDIX  143 

ment.  The  head  occupies  different  positions.  In  some  it  is  drawn 
back  as  in  tetanus ;  in  others  it  is  drawn  down  with  the  nose  near 
the  fore  legs ;  and  in  still  others  it  is  extended  as  if  the  animal  were 
sleeping.  The  period  of  this  complete  paralysis  varies  from  a  few 
hours  to  a  few  days,  but  ordinarily  it  does  not  exceed  24  hours. 
Although  the  animals  are  unable  to  move  voluntarily,  there  is  usually 
a  reflex  action  of  the  limbs  until  a  very  short  time  before  death. 

During  the  period  of  incubation  the  temperature  of  the  rabbits 
remains  normal.  As  the  time  approaches  for  the  first  symptoms  to 
appear  there  has  been  in  the  animals  tested  an  elevation  of  tem- 
perature of  from  I  to  2  degrees,  which  continued  for  a  variable 
length  of  time,  but  rarely  longer  than  2  days.  This  is  followed 
by  a  gradual  or  usually  a  more  rapid  drop  to  the  subnormal,  which 
continues  to  the  end.  j> 

Swine  Plague.  —  Rabbits  are  most  susceptible.  Inoculate  sud- 
cutaneously  with  a  bit  of  the  pneumonic  tissue,  either  in  a  solid 
piece  or  in  a  suspension  in  bouillon  hypodermically.  In  case  of 
virulent  swine-plague  bacteria,  the  rabbit  will  die  in  from  16  to 
36  hours  from  septicaemia.  Pure  cultures  can  be  obtained  from 
the  blood,  spleen,  liver,  or  kidney.  Stained  cover-glass  prepara- 
tions from  these  organs  show  a  greater  or  less  number  of  polar- 
stained  bacteria. 

In  case  of  a  more  attenuated  virus  the  rabbit  will  live  from  a  few 
days  to  several  weeks  and  possibly  months.  In  these  cases  there 
are  usually  marked  local  cell  infiltrations,  with  inflammation  of  one 
or  more  of  the  serous  membranes  and  possibly  metastatic  abscesses. 

Hog  Cholera.  —  Rabbits  are  most  desirable.  They  are  inoculated 
in  the  same  manner  as  with  tuberculosis.  With  ordinarily  virulent 
bacteria  the  rabbits  will  die  in  from  seven  to  ten  days.  The  lesions 
are  essentially  a  purulent  infiltration  of  the  subcutis  at  the  point  of 
inoculation,  an  enlarged  and  very  dark  colored  spleen,  and  areas  of 
coagulation  necrosis  in  the  liver.  Pure  cultures  can  be  obtained 
from  the  blood,  liver,  or  spleen. 

Antirax.  —  Mice  or  guinea  pigs  should  be  used.  They  are  inocu- 
lated in  the  manner  described  above.  They  die  of  septicaemia  usu- 
ally in  from  24  to  72  hours.  It  is  not  commonly  necessary  to 
resort  to  animal  inoculation  with  this  disease.  Occasionally,  how- 
ever, it  is  a  very  necessary  procedure  in  making  an  early  diagnosis. 

Diphtheria.  —  Guinea  pigs  are  nearly  always  used.     In  certain  rare 


•144  LABORATORY   BACTERIOLOGY 

cases  of  mixed  c.ultures  taken  directly  from  the  suspected  throat  it 
is  desirable  to  inoculate  one  or  more  guinea  pigs  to  determine 
whether  the  suspected  organism  present  is  a  virulent  Klebs-Loeffler 
bacterium.  In  these  cases  a  suspension  of  the  growth  on  the  serum 
may  be  injected.  The  guinea  pig  dies  usually  in  from  36  to  80 
hours.  The  lesions  produced  have  been  described  by  Park  as 
follows : 

^^  At  the  seat  of  inoculation  there  is  a  grayish  focus  surrounded  by 
an  area  of  congestion ;  the  subcutaneous  tissues  for  some  distance 
around  are  oedematous  ;  the  adjacent  lymph  nodes  are  swollen  ;  and 
the  serous  cavities,  especially  the  pleural  and  the  pericardial,  fre- 
quently contain  an  excess  of  fluid,  usually  clear,  but  at  times  turbid  ; 
the  lungs  are  generally  congested.  In  the  organs  are  found  numer- 
ous smaller  and  larger  masses  of  necrotic  cells,  which  are  permeated 
by  leucocytes.  The  heart  and  voluntary  muscle  fibres  usually  show 
degenerative  changes.  Occasionally  there  is  fatty  degeneration  of 
the  liver  and  kidneys.  The  number  of  leucocytes  in  the  blood  is 
increased.  From  the  area  surrounding  the  point  of  inoculation,  vir- 
ulent bacilli  may  be  obtained,  but  in  the  internal  organs  they  are 
only  occasionally  found,  unless  an  enormous  number  of  bacilli  have 
been  injected.  Paralysis,  commencing  usually  in  the  posterior  ex- 
tremities, and  then  gradually  extending  to  other  portions  of  the  body 
and  causing  death  by  paralysis  of  the  heart  or  respiratory  organs, 
is  also  produced  in  many  cases  in  which  the  inoculated  animals  do 
not  succumb  to  a  too  rapid  intoxication.'' 

Guinea  pigs  are  used  for  testing  the  virulence  of  ]Dure  cultures  and 
the  strength  of  the  toxin  and  antitoxin.  For  further  details,  see 
text-books  of  bacteriology. 


APPENDIX 


MS 


IV 

CULTIVATION   OF    BACTERIUM    (BACILLUS)    TUBERCULOSIS 

The  isolation  of  this  organism  from  tuberculous  lesions  and  get- 
ting it  to  multiply  readily  on  artificial  media  necessitates  a  very 
special  and  careful  procedure.  When  it  becomes  accustomed  to 
artificial  media  its  continued  cultivation  is  not  difficult.  Dr.  Theo- 
bald Smith,  of  Harvard  University  (Jour,  of  Exp.  Med.,  Vol.  IIL, 
1898,  p.  451),  has  the  credit  of  formulating  a  method  by  combining 
details  in  such  a  manner  that  the  procuring  of  cultures  is,  in  most 
cases,  possible.  Dog  serum  is  used.  The  method,  as  he  gives  it, 
is  as  follows,  viz. : 

"  The  dog  was  bled  under  chloroform  and  the  blood  drawn  from 
a  femoral  artery,  under  aseptic  conditions,  through  sterile  tubes 
directly  into  sterile  flasks.  The  serum  was  drawn  from  the  clots 
with  sterile  pipettes  and  either  distributed  at  once  into  tubes  or  else 
stored  with  0.25  to  0.3%  chloroform  added.  Discontinued  ster- 
ilization was  rendered  unnecessary.  The  temperature  required  to 
produce  a  sufficiently  firm  and  yet  not  too  hard  and  dry  serum  is 
for  the  dog  75°  to  76^  C.  For  horse  serum  it  is  from  4°  to  5°  lower. 
The  serum  was  set  in  a  thermostat  into  which  a  large  dish  of  water 
was  always  placed  to  forestall  any  abstraction  of  moisture  from  the 
serum.  About  3  hours  suffice  for  the  coagulation.  When  serum 
containing  chloroform  is  to  be  coagulated,  I  am  in  the  habit  of 
placing  the  tubes  for  an  hour  or  longer  in  a  water  bath  at  55°  to  60° 
C,  or  under  the  receiver  of  an  air  pump,  to  drive  off  the  antiseptic. 
This  procedure  dispenses  with  all  sterilization  excepting  that  going 
on  during  the  coagulation  of  the  serum.  It  prevents  the  gradual 
formation  of  membranes  of  salts,  which,  remaining  on  the  surface 
during  coagulation,  form  a  film  unsuited  for  bacteria.  Tubes  of 
coagulated  serum  should  be  kept  in  a  cold  closed  space  where  the 
opportunities  for  evaporation  are  slight.  They- should  always  be 
kept  inclined. 

The  ordinary  cotton-plugged  test  tubes  I  do  not  use,  because  of 
the  rapid  drying  out  permitted  by  them,  as  well  as  the  opportunities 
for  infection  with  fungi.  Instead,  a  tube  is  used  which  has  a  ground 
glass   cap  fitted  over  it.     This   cap  contracts   into  a  narrow  tube 


146  LABORATORY  BACTERIOLOGY 

plugged  with  glass  wool.  This  plug  is  not  disturbed.  The  tube  is 
cleaned,  filled,  and  inoculated  by  removing  the  cap.  With  sufficient 
opportunity  for  the  interchange  of  air  little  evaporation  takes  place, 
and  contamination  of  the  culture  is  of  very  rare  occurrence.  In  inoc- 
ulating these  tubes,  bits  of  tissue,  which  include  tuberculous  foci, 
especially  the  most  recent,  are  torn  from  the  organs  and  transferred 
to  the  serum.  Very  little  crushing,  if  any,  is  desirable  or  necessary. 
I  think  many  failures  are  due  to  the  often  futile  attempts  to  break 
up  firm  tubercules.  Nor  should  the  bits  of  tissue  be  rubbed  into 
the  surface,  as  is  sometimes  recommended.  After  a  stay  of  several 
weeks  in  the  thermostat,  I  usually  remove  the  tubes  and  stir  about 
the  bits  of  tissue.  This  frequently  is  the  occasion  for  a  prompt 
appearance  of  growth  within  a  week,  as  it  seems  to  put  certain  still 
microscopic  colonies  in  or  around  the  tissue  into  better  condition  for 
further  development.  The  thermostat  should  be  fairly  constant,  as 
urged  by  Koch  in  his  classic  monograph,  but  I  look  upon  moisture 
as  more  important.  If  possible,  a  thermostat  should  be  used  which 
is  opened  only  occasionally.  Into  this  a  large  dish  of  water  is 
placed,  which  keeps  the  space  saturated.  Ventilation  should  be 
restricted  to  a  minimum.  As  a  consequence,  moulds  grow  luxuri- 
antly and  even  the  gummed  labels  must  be  replaced  by  pieces  of 
stiff  manila  paper  fastened  to  the  tube  with  a  rubber  band.  By 
keeping  the  tubes  inclined,  no  undue  amount  of  condensation  water 
can  collect  in  the  bottom,  and  the  upper  portion  of  the  serum 
remains  moist.  The  only  precaution  to  be  applied  to  prevent  infec- 
tion with  moulds  is  to  thoroughly  flame  the  joint  between  tube  and 
cap  as  well  as  the  plugged  end,  before  opening  the  tube.  When 
test  tubes  are  employed  it  is  well  to  dip  the  lower  end  of  the  plug 
into  sterile  molten  paraffin  and  to  cover  the  tube  with  a  sterilized 
paper  cap.  The  white  bottle  caps  of  the  druggist  are  very  service- 
able." 

Unless  the  tuberculous  material  is  perfectly  fresh  (uncontami- 
nated),  and  in  the  early  stages  of  the  disease,  it  is  safer  to  inoculate  a 
guinea  pig,  and  after  the  lesions  begin  to  develop  to  chloroform  it 
and  make  the  cultures  from  the  recently  affected  liver  or  spleen. 


JEFFERS    PLATE 
For  Counting  Colonies  of  Bacteria  in  Petri  Dishes. 

The  area  of  each  division  is  one  square  centimeter. 


THE    METRIC    SYSTEM. 


^I^HKI^^Sl^^M^^ri^^^^ 


THE  METER  FOR 
LENGTH     .    .    . 


10  CENTIMETER  RULE. 

The  upper  edge  is  in  millimeters,  the  lower  in  centimeter  and  half  centimeters. 
UNITS.  The  most  commonly  used  divisions  and  multiples. 

Centimeter  (cm.),  i/iooth  meter;    millimeter  (mm.), 
i/ioooth  meter;  micron  {fi),  i/ioooth  millimeter; 
the  micron  is  the  unit  in  Micrometry. 
Kilometer  J   I  coo  meters  ;    used  in   measuring  roads 
and  other  long  distances. 
THE   GRAM    FOR  (  Milligram  (mg,),  i/roooth  gram. 

WEIGHT    ...  I  Kilogram^  looo  grams;  used  for  ordinary  masses. 
THE  LITER  FOR  j  Cubic  Centimeter  (cc),  i/ioooth  liter.     This  is  more 
CAPACITY    .  .  I      common  than  the  correct  form,  milliliter. 
Divisions  of  the  Units  are  indicated    by  the  Latin  prefixes  :    deci^ 
l/ioth  ;  centiy  i/iooth;  milli,  i/ioooth. 

Multiples  are  designated  by  Greek  prefixes:    deka,  lo  times;  /lecto^ 
100  times;  kilo^  looo  times  ;  myria^  10,000  times. 

Table  of  Metric  and  English  Measures. 

Meters  =  100  centimeters,  1000  millimeters,  1,000,000  yot,  393704  inches. 
Millimeter  (mm.)  =  1000  microns,  i/ioth  millimeter,  i/i 000th  meter, 

I /25th  inch,  approximately. 
Micron     (/x)     (Unit    of    Measure     in    Micrometry)   =  i/ioooth   mm., 

i/iooooooth  meter  (0.000039  inch),  1/2 5000th  inch,  approximately. 
Inch  (in.)  =  25.399772  mm.  (25.4  mm.,  approx.). 

Liter  =  1000  milliliters  or  1000  cubic   centimeters,  i  quart  (approx.). 
Cubic  centimeter  (cc.  or  cctm.)  =  i/ioooth  of  a  liter. 
Fluid  ounce  (8  fluidrachms)  =  29.578  cc.  (30  cc,  approx.). 
Gram  =  15.432  grains. 

Kilogram  (kilo)  =  2.204  avoirdupois  pounds  (2  i/5th  pounds,  approx.). 
Ounce  avoirdupois  =  (437  1/2  grains)  ==  28.349  grams. 
Ounce  troy  or  apothecary's  =  (480  grains)  =  31.103  grams. 

Temperature. 

To  change  Centigrade  to  Fahrenheit:  (C.  x  9/5)  +  32  =  F.  For 
example,  to  find  the  equivalent  of  10°  Centigrade,  C.  =  10'' 
(10°  X  9/5)  +  32  =  50°  F. 
To  change  Fahrenheit  to  Centigrade  :  (F.  —  32^)  x  5/9  =  C.  For 
example,  to  reduce  50°  Fahrenheit  to  Centigrade,  F.  =  50°,  and 
(50°  "  32°)  X  5/9=  10°  C;  or  —40  Fahrenheit  to  Centigrade, 
F.  =  -  40°  (-  40°  -  32°)  -  -  72°  whence  -  72°  x  5/9  =  -40°C. 
(^Frofft  ^^The  Microscope,''^  by  Fro/.  S .  H.  Gage,  used  here  with  his  per  mission.) 

148 


INDEX 


Agar,  7,  ii. 

acid,  42. 

acid  glycerin,  42. 

glycerin,  41. 

glucose,  41. 

inoculating  tubes  of,  15. 

preparation  of,  12. 
Anaerobic  bacteria,  96. 

cultivation  of,  96. 

fermentation  tube  for,  97. 

Liborius'  method,  97. 
Aniline  water,  28. 
Animal  inoculation  for  diagnosis, 
140. 

methods,  140. 

for  anthrax,  143. 

for  diphtheria,  143. 

for  glanders,  141. 

for  hog  cholera,  143. 

for  rabies,  141. 

for  swine  plague,  143. 

for  tuberculosis,  141. 
Apparatus.     See  Glassware. 
Autoclave,  use  of,  9. 

Bacillus,  morphology  of,  58. 

cholerae  suis,  79. 

coli  communis,  76. 

of  dysentery,  84. 

tetani,  98. 

typhosus,  79. 
Bacteria,  classification  of,  53. 

drawings  of,  59. 

identifying  species,  69,  107. 

in  air,  67. 


in  milk,  71. 
in  mouth,  105. 
in  tissues,  108. 
in  skin,  114. 
in  water,  72. 
determining  genera,  69. 
study  of  certain  species,  126. 
pyogenic,  73. 

,  gas-producing,  in  water,  estimat- 
ing number,  125. 
Bacteriological  diagnosis,  127. 
Bacterium,  morphology  of,  59. 
anthracis,  100. 
diphtheriae,  102. 
mallei,  94. 

septicaemiae  hemorrhagicae,  88. 
tuberculosis,  67,  91. 
Blood  serum,  42. 

Loeffler's,  43. 
Bouillon,  preparation  of,  8. 

acid  glycerin,  42. 

glucose,  41. 

lactose,  41. 

nitrate,  43. 

saccharose,  41. 

titration,  10. 
inoculating,  14. 
sugar-free,  41. 

Chester's  terminology,  20,  34. 
Cleaning  mixture,  formula,  2. 
Colonies,  estimating  number,  ^^. 

subcultures  from,  34. 
Cotton,  absorbent,  5. 

common,  5. 


149 


THE   METRIC    SYSTEM. 


^  I^^Ki^^n^^K|^^:i^^» 


0 


THE  METER  FOR 
LENGTH     .    .    . 


10  CENTIMETER  RULE. 

The  upper  edge  is  in  millimeters,  the  lower  in  centimeter  and  half  centimeters. 
UNITS.  The  most  commonly  used  divisions  and  multiples. 

Centimeter  (cm.),  i/iooth  meter;    millimeter  (mm.), 
i/ioooth  meter;  micron  (fx),  i/ioooth  millimeter; 
the  micron  is  the  unit  in  Micrometry. 
Kilometer^   looo  meters  ;    used  in   measuring  roads 
and  other  long  distances. 
THE   GRAM    FOR  j  Milligram  (mg.),  i/ioooth  gram. 

WEIGHT    .  .  .  (  Kilogramy  looo  grams;  used  for  ordinary  masses. 
THE   LITER  FOR  (  Cubic  centimeter  (cc),  i/ioooth  liter.     This  is  more 
CAPACITY    .  .  \      common  than  the  correct  form,  milliliter. 
Divisions  of  the  Units  are  indicated    by  the  Latin  prefixes  :    deci^ 
l/ioth  ;  centi^  i/iooth;  milli^  i/ioooth. 

Multiples  are  designated  by  Greek  prefixes:    deka,  lo  times;  hecto^ 
100  times;  kiloy  looo  times  ;  myria,  io,ooo  times. 

Table  of  Metric  and  English  Measures. 

Meters  =  loo  centimeters,  looo  millimeters,  1,000,000  /x,  39.3704  inches. 
Millimeter  (mm.)  =  1000  microns,  i/ioth  millimeter,   i/ioooth  meter, 

I /25th  inch,  approximately. 
Micron     (/x)     (Unit    of    Measure     in     Micrometry)    =  i/ioooth    mm., 

i/iooooooth  meter  (0.000039  inch),  1/2 5000th  inch,  approximately. 
Inch  (in.)  =  25.399772  mm.  (25.4  mm.,  approx.). 

Liter  =  1000  milliliters  or  1000  cubic   centimeters,  i  quart  (approx.). 
Cubic  centimeter  (cc.  or  cctm.)  =  i/ioooth  of  a  liter. 
Fluid  ounce  (8  fluidrachms)  =  29.578  cc.  (30  cc,  approx.). 
Gram  =  15.432  grains. 

Kilogram  (kilo)  =  2.204  avoirdupois  pounds  (2  i/5th  pounds,  approx.). 
Ounce  avoirdupois  =  (437  1/2  grains)  —  28.349  grams. 
Ounce  troy  or  apothecary's  =  (480  grains)  =  31-103  grams. 

Temperature. 

To  change  Centigrade  to  Fahrenheit :  (C.  x  9/5)  +  32  =  F.  For 
example,  to  find  the  equivalent  of  10°  Centigrade,  C.  =  lo'' 
(10°  X  9/5)  -f  32  z=  50°  F. 

To  change  Fahrenheit  to  Centigrade  :  (F.  —  32")  x  5/9  =  C.  For 
example,  to  reduce  50°  Fahrenheit  to  Centigrade,  F.  =  50°,  and 
(50°  ~  3-°)  X  5/9=  'o°  C.;  or  —40  Fahrenheit  to  Centigrade, 
F.  =  -  40°  (-  40°  -  i,!"")  =  -  72°,  whence  -  72°  x  5/9  =  -40°  C. 


{From  ^''The  Microscope,^ 


by  Prof.  S .  H.  Gagey  used  here  with  his  permission.) 
148 


INDEX 


Agar,  7,  II. 

acid,  42. 

acid  glycerin,  42. 

glycerin,  41. 

glucose,  41. 

inoculating  tubes  of,  15. 

preparation  of,  12. 
Anaerobic  bacteria,  96. 

cultivation  of,  96. 

fermentation  tube  for,  97. 

Liborius'  method,  97. 
Aniline  water,  28. 
Animal  inoculation  for  diagnosis, 
140. 

methods,  140. 

for  anthrax,  143. 

for  diphtheria,  143. 

for  glanders,  141. 

for  hog  cholera,  143. 

for  rabies,  141. 

for  swine  plague,  143. 

for  tuberculosis,  141. 
Apparatus.     See  Glassware. 
Autoclave,  use  of,  9. 

Bacillus,  morphology  of,  58. 

cholerae  suis,  79. 

coli  communis,  76. 

of  dysentery,  84. 

tetani,  98. 

typhosus,  79. 
Bacteria,  classification  of,  53. 

drawings  of,  59. 

identifying  species,  69,  107. 


67. 


in  milk,  71. 
in  mouth,  105. 
in  tissues,  108. 
in  skin,  114. 
in  water,  72. 
determining  genera,  69. 
study  of  certain  species,  126. 
pyogenic,  73. 

.  gas-producing,  in  water,  estimat- 
ing number,  125. 
Bacteriological  diagnosis,  127. 
Bacterium,  morphology  of,  59. 
anthracis,  100. 
diphtheriae,  102. 
mallei,  94. 

septicaemias  hemorrhagicae,  88. 
tuberculosis,  67,  91. 
Blood  serum,  42. 

Loeffler's,  43. 
Bouillon,  preparation  of,  8. 

acid  glycerin,  42. 

glucose,  41. 

lactose,  41. 

nitrate,  43. 

saccharose,  41. 

titration,  10. 
inoculating,  14. 
sugar-free,  41. 

Chester's  terminology,  20,  34. 
Cleaning  mixture,  formula,  2. 
Colonies,  estimating  number,  ^'^. 

subcultures  from,  34. 
Cotton,  absorbent,  5. 

common,  5. 


149 


ISO 


INDEX 


Cover  glasses,  cleaning  of,  2. 

for  flagella  stain,  3. 
Cover-glass  preparations,  23. 

from  blood,  82. 

from  milk,  52. 

from  pus,  113. 

from  sputum,  92. 

from  tissues,  81. 
Cultivation  of  Bact.  tuberculosis, 

145. 
Cultures,     examination     of,     17, 

48. 

anaerobic,  96. 

from  tissues,  109. 

hanging-drop  preparations,  19. 

labeling,  10. 

macroscopic  examination  of,  1 7, 

49. 
microscopic  examination  of,  19, 

25- 
reaction  of  liquid,  18. 
sealing,  16. 
viscidity  of,  18. 

Disinfectants,  testing  efficiency  of, 

118. 
Drawings  of  bacteria,  59. 
Dunham's  solution,  77. 


Gabbett's  method,  68,  93. 
Gas,  production  by  bacteria,  50 

quantity  produced,  50. 

ratio  of  CO2  to  H,  50. 
Gelatin,  7,  11. 

inoculating  tubes  of,  16. 

preparation  of,  12. 
Genera,  identifying,  67. 
Glassware,  i. 

cleaning  of,  1-3. 

sterilizing,  6. 
Gram's  method  of  staining,  28. 

Identifying  bacteria  from  tissues, 

108. 
Indol  test,  77. 
Inoculating  media,  14,  46. 
Isolating  B.  coli  communis  from 

intestine,  78. 

Jeffers'  plate,  ^^^  147. 

Labeling  media  and  cultures,  10. 

cover-glass  preparations,  25. 
Laboratory  notes,  4. 
Liborius'  method,   anaerobic  cul- 
tures, 97. 
Lugol's  solution,  28. 


Egg  as  a  culture  medium,  43. 
Exudates,    microscopic    examina- 
tion of,   112. 

Fermentation  tube,  48,  50,  97. 
Filar  micrometer,  137. 
Flagella,  58. 

staining  of,  62. 

Johnston's  and  Mack's  method, 

63- 

Loeffler's  method,  65. 

Van  Ermengem's  method,  66. 


Media,  reaction  of,  131. 

neutralization,  134. 

preparation  of,  39. 

grouping  of,  43. 

short  method  of  sterilizing,  9. 

special,  39. 

effect  of  bacteria  upon,  48. 
Metric  system,  148. 
Micrococcus,  morphology  of,  56. 

epidermidis  albus,  114. 

lanceolatus,  90. 

pyogenes  aureus,  73. 


INDEX 


151 


Micrometer,  eye-piece,  136. 

measurements,  138. 

ocular,  136. 

valuation  of,  136,  138. 

varying  valuation  of,  139. 
Micrometry,  136. 
Microscopic    examinations.      See 

Culture. 
Migula's  classification  of  bacteria, 

54. 
Milk  as  a  culture  medium,  40. 

litmus,  40. 
Mouth,  bacteria  in,  105. 

making  cover-glass  preparations 
from,  106. 

Neisser's  method  of  staining,  103. 

Paracolon,  78. 
Pasteurization  of  milk,  1 20. 
Plate  cultures,  use  of,  29. 

agar,  30. 

examination  of,  32. 

gelatin,  30. 
Plugging  tubes  and  flasks,  5. 
Potato  as  a  culture  medium,  40. 
Pseudomonas  pyocyaneus,  75. 
Pus,  examination  of,  112. 

Roll  cultures,  Esmarch,  31. 
examination  of,  32. 


Staining  solutions,  25. 

formulas,  26. 

actinomyces,  127. 

Gram's  solution,  28. 

alkaline  methylene  blue,  26. 

aniline  gentian  violet,  27. 

aqueous  solutions,  27. 

carbol  fuchsin,  26. 

carbolic  thionine  blue,  27. 

carbolic  gentian  violet,  27. 

malarial  parasites,  127. 

Sudan  III,  93. 
Staining  spores,  60. 
Stains,  23. 

Staphylococcus.    See  Micrococcus. 
Sterilizer,  use  of  hot  air,  5. 
Sterilizing  milk,  120. 
Streptococcus,  morphology  of,  56. 
Subcultures,  making  of,  34. 
Sudan  III,  93. 
Sugars,  use  of  media  containing, 

46. 

Temperature,  centigrade,  148. 

Fahrenheit,  148. 
Tetanus,  isolating  bacilli  of,  98. 

Kitasato's  method,  99. 
Thermal  death  point  of  bacteria, 

116. 
Tubercle  bacteria,  67. 

staining  of,  68. 


Saprophytic  bacteria,  69. 
Sarcina,  morphology  of,  56. 
Spirillum,  morphology  of,  59. 
Spores,  60. 

method  of  staining,  61. 
Sputum,  92. 

cover-glass  preparation,  92. 
Staining  bacteria,  24. 

flagella,  62. 


Water,  bacteriologic  examination 
of,  122. 

qualitative,  124. 

quantitative,  122. 

collecting,  123. 
Widal  serum  test,  85. 

securing  blood  for,  86. 
Wolffhiigel's  counting  apparatus, 


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